Transgenic animals expressing heparanase and uses thereof

ABSTRACT

A transgenic non-human animal expressing heparanase from a transgene, methods for its preparation, compositions-of-matter derived therefrom and uses thereof.

[0001] This is a continuation-in-part of U.S. patent application Ser.No. 09/988,113, filed Feb. 6, 2001, which is a continuation of U.S.patent application Ser. No. 09/776,874, filed Feb. 6, 2001, which is acontinuation of U.S. patent application Ser. No. 09/258,892, filed Mar.1, 1999, now abandoned, which is a continuation-in-part ofPCT/US98/17954, filed Aug. 31, 1998, which claims priority from U.S.patent application Ser. No. 09/109,386, filed Jul. 2, 1998, nowabandoned, which is a continuation-in-part of U.S. patent applicationSer. No. 08/922,170, filed Sep. 2, 1997, now, U.S. Pat. No. 5,968,822,issued Oct. 19, 1999. This application is also a continuation-in-part ofU.S. patent application Ser. No. 09/864,321, filed May 25, 2001.

FIELD AND BACKGROUND OF THE INVENTION

[0002] The present invention relates to transgenic animals expressingheparanase and to the uses thereof as a model for human disease and forthe commercial production of heparanase.

[0003] Glycosaminoglycans (GAGs):

[0004] GAGs are polymers of repeated disaccharide units consisting ofuronic acid and a hexosamine. Biosynthesis of GAGs except hyaluronicacid is initiated from a core protein. Proteoglycans may contain severalGAG side chains from similar or different families. GAGs are synthesizedas homopolymers which may subsequently be modified by N-deacetylationand N-sulfation, followed by C5-epimerization of glucuronic acid toiduronic acid and O-sulfation. The chemical composition of GAGs fromvarious tissues varies to a great extent.

[0005] The natural metabolism of GAGs in animals is carried out byhydrolysis. Generally, the GAGs are degraded in a two step procedure.First the proteoglycans are internalized in endosomes, where initialdepolymerization of the GAG chain takes place. This step is mainlyhydrolytic and yields oligosaccharides. Further degradation is carriedout following fusion with lysosome, where desulfation and exolyticdepolymerization to monosaccharides take place (42).

[0006] The only GAG degrading endolytic enzymes characterized so far inanimals are the hyaluronidases. The hyaluronidases are a family of 1-4endoglucosaminidases that depolymerize hyaluronic acid and chondroitinsulfate. The cDNAs encoding sperm associated PH-20 (Hyal3), and thelysosomal hyaluronidases Hyal 1 and Hyal 2 were cloned and published(27). These enzymes share an overall homology of 40% and have differenttissue specificities, cellular localizations and pH optima for activity.

[0007] Exolytic hydrolases are better characterized, among which arebeta-glucuronidase, alpha-L-iduronidase andbeta-N-acetylglucosaminidase. In addition to hydrolysis of theglycosidic bond of the polysaccharide chain, GAG degradation involvesdesulfation, which is catalyzed by several lysosomal sulfatases such asN-acetylgalactosamine-4-sulfatase, iduronate-2-sulfatase and heparinsulfamidase. Deficiency in any of lysosomal GAG degrading enzymesresults in a lysosomal storage disease known as mucopolysaccharidosis.

[0008] Glycosyl Hydrolases:

[0009] Glycosyl hydrolases are a widespread group of enzymes thathydrolyze the o-glycosidic bond between two or more carbohydrates orbetween a carbohydrate and a noncarbohydrate moiety. The enzymatichydrolysis of glycosidic bond occurs by one or two major mechanismsleading to overall retention or inversion of the anomeric configuration.In both mechanisms, catalysis involves a proton donor and a nucleophile.Glycosyl hydrolyses have been classified into 58 families based on aminoacid similarities. The glycosyl hydrolyses from families 1, 2, 5, 10,17, 30, 35, 39 and 42 act on a large variety of substrates, however,they all hydrolyze the glycosidic bond in a general acid catalysismechanism, with retention of the anomeric configuration. The mechanisminvolves two glutamic acid residues, which serve as the proton donor andthe nucleophile, with an asparagine, which always precedes the protondonor. Analyses of a set of known 3D structures from this group ofenzymes revealed that their catalytic domains, despite the low level ofsequence identity, adopt a similar (alpha/beta) 8 fold with the protondonor and the nucleophile located at the C-terminal ends of strands beta4 and beta 7, respectively. Mutations in the functional conserved aminoacids of lysosomal glycosyl hydrolases were identified in lysosomalstorage diseases.

[0010] Lysosomal glycosyl hydrolases including beta-glucuronidase,beta-mannosidase, beta-glucocerebrosidase, beta-galactosidase andalpha-L-iduronidase, are all exo-glycosyl hydrolases, belong to the GH-Aclan and share a similar catalytic site. However, many endo-glucanasesfrom various organisms, such as bacterial and fungal xylenases andcellulases share this catalytic domain (1).

[0011] Heparan Sulfate Proteoglycans (HSPGs):

[0012] HSPGs are ubiquitous macromolecules associated with the cellsurface and extracellular matrix (ECM) of a wide range of cells ofvertebrate and invertebrate tissues (3-7). The basic HSPG structureconsists of a protein core to which several linear heparan sulfatechains are covalently attached. The polysaccharide chains are typicallycomposed of repeating hexuronic and D-glucosamine disaccharide unitsthat are substituted to a varying extent with N- and O-linked sulfatemoieties and N-linked acetyl groups (3-7). Studies on the involvement ofECM molecules in cell attachment, growth and differentiation revealed acentral role of HSPGs in embryonic morphogenesis, angiogenesis,metastasis, neurite outgrowth and tissue repair (3-7). The heparansulfate (HS) chains, which are unique in their ability to bind amultitude of proteins, ensure that a wide variety of effector moleculescling to the cell surface (6-8). HSPGs are also prominent components ofblood vessels (5). In large vessels they are concentrated mostly in theintima and inner media, whereas in capillaries they are found mainly inthe subendothelial basement membrane where they support proliferatingand migrating endothelial cells and stabilize the structure of thecapillary wall. The ability of HSPGs to interact with ECM macromoleculessuch as collagen, laminin and fibronectin, and with different attachmentsites on plasma membranes suggests a key role for this proteoglycan inthe self-assembly and insolubility of ECM components, as well as in celladhesion and locomotion. Cleavage of HS may therefore result indisassembly of the subendothelial ECM and hence may play a decisive rolein extravasation of normal and malignant blood-borne cells (9-11). HScatabolism is observed in inflammation, wound repair, diabetes, andcancer metastasis, suggesting that enzymes, which degrade HS, playimportant roles in pathologic processes.

[0013] Heparanase:

[0014] Heparanase is a glycosylated enzyme that is involved in thecatabolism of certain glycosaminoglycans. It is an endoglucuronidasethat cleaves heparan sulfate at specific intrachain sites (12-15).Interaction of T and B lymphocytes, platelets, granulocytes, macrophagesand mast cells with the subendothelial extracellular matrix (ECM) isassociated with degradation of heparan sulfate by heparanase activity(16). Placental heparanase acts as an adhesion molecule or as adegradative enzyme depending on the pH of the microenvironment (17).

[0015] Heparanase is released from intracellular compartments (e.g.,lysosomes, specific granules) in response to various activation signals(e.g., thrombin, calcium ionophores, immune complexes, antigens andmitogens), suggesting its regulated involvement in inflammation andcellular immunity responses (16).

[0016] It was also demonstrated that heparanase can be readily releasedfrom human neutrophils by 60 minutes incubation at 4° C. in the absenceof added stimuli (18).

[0017] Gelatinase, another ECM degrading enzyme, which is found intertiary granules of human neutrophils with heparanase, is secreted fromthe neutrophils in response to phorbol 12-myristate 13-acetate (PMA)treatment (19-20).

[0018] In contrast, various tumor cells appear to express and secreteheparanase in a constitutive manner in correlation with their metastaticpotential (21).

[0019] Degradation of heparan sulfate by heparanase results in therelease of heparin-binding growth factors, enzymes and plasma proteinsthat are sequestered by heparan sulfate in basement membranes,extracellular matrices and cell surfaces (22-23).

[0020] Heparanase activity has been described in a number of cell typesincluding cultured skin fibroblasts, human neutrophils, activated ratT-lymphocytes, normal and neoplastic murine B-lymphocytes, humanmonocytes and human umbilical vein endothelial cells, SK hepatoma cells,human placenta and human platelets.

[0021] Procedures for purification of natural heparanase were reportedfor SK hepatoma cells and human placenta (U.S. Pat. No. 5,362,641) andfor human platelets derived enzymes (53).

[0022] Involvement of Heparanase in Tumor Cell Invasion and Metastasis:

[0023] Circulating tumor cells arrested in the capillary beds oftenattach at or near the intercellular junctions between adjacentendothelial cells. Such attachment of the metastatic cells is followedby rupture of the junctions, retraction of the endothelial cell bordersand migration through the breach in the endothelium toward the exposedunderlying base membrane (BM) (24). Once located between endothelialcells and the BM, the invading cells must degrade the subendothelialglycoproteins and proteoglycans of the BM in order to migrate out of thevascular compartment. Several cellular enzymes (e.g., collagenase IV,plasminogen activator, cathepsin B, elastase, etc.) are thought to beinvolved in degradation of BM (25). Among these enzymes is heparanasethat cleaves HS at specific intrachain sites (16, 11). Expression of aHS degrading heparanase was found to correlate with the metastaticpotential of mouse lymphoma (26), fibrosarcoma and melanoma (21) cells.Moreover, elevated levels of heparanase were detected in sera frommetastatic tumor bearing animals and melanoma patients (21) and in tumorbiopsies of cancer patients (12).

[0024] The inhibitory effect of various non-anticoagulant species ofheparin on heparanase was examined in view of their potential use inpreventing extravasation of blood-borne cells. Treatment of experimentalanimals with heparanase inhibitors markedly reduced (>90%) the incidenceof lung metastases induced by B16 melanoma, Lewis lung carcinoma andmammary adenocarcinoma cells (12, 13, 28). Heparin fractions with highand low affinity to anti-thrombin III exhibited a comparable highanti-metastatic activity, indicating that the heparanase inhibitingactivity of heparin, rather than its anticoagulant activity, plays arole in the anti-metastatic properties of the polysaccharide (12).

[0025] The direct role of heparanase in cancer metastasis wasdemonstrated by two experimental systems. The murine T-lymphoma cellline Eb has no detectable heparanase activity. Whether the introductionof the hpa gene into Eb cells would confer a metastatic behavior onthese cells was investigated. To this purpose, Eb cells were transfectedwith a full length human hpa cDNA. Stable transfected cells showed highexpression of the heparanase mRNA and enzyme activity. These hpa andmock transfected Eb cells were injected subcutaneously into DBA/2 miceand mice were tested for survival time and liver metastases. All mice(n=20) injected with mock transfected cells survived during the first 4weeks of the experiment, while 50% mortality was observed in miceinoculated with Eb cells transfected with the hpa cDNA. The liver ofmice inoculated with hpa transfected cells was infiltrated with numerousEb lymphoma cells, as was evident both by macroscopic evaluation of theliver surface and microscopic examination of tissue sections. Incontrast, metastatic lesions could not be detected by gross examinationof the liver of mice inoculated with mock transfected control Eb cells.Few or no lymphoma cells were found to infiltrate the liver tissue. In adifferent model of tumor metastasis, transient transfection of theheparanase gene into low metastatic B16-F1 mouse melanoma cells followedby intravenous inoculation, resulted in a 4- to 5-fold increase in lungmetastases.

[0026] Finally, heparanase externally adhered to B16-F1 melanoma cellsincreased the level of lung metastases in C57BL mice as compared tocontrol mice (see U.S. patent application Ser. No. 09/260,037, which isincorporated herein by reference).

[0027] Possible Involvement of Heparanase in Tumor Angiogenesis:

[0028] Fibroblast growth factors are a family of structurally relatedpolypeptides characterized by high affinity to heparin (29). They arehighly mitogenic for vascular endothelial cells and are among the mostpotent inducers of neovascularization (29-30). Basic fibroblast growthfactor (bFGF) has been extracted from a subendothelial ECM produced invitro (31) and from basement membranes of the cornea (32), suggestingthat ECM may serve as a reservoir for bFGF. Immunohistochemical stainingrevealed the localization of bFGF in basement membranes of diversetissues and blood vessels (23). Despite the ubiquitous presence of bFGFin normal tissues, endothelial cell proliferation in these tissues isusually very low, suggesting that bFGF is somehow sequestered from itssite of action. Studies on the interaction of bFGF with ECM revealedthat bFGF binds to HSPG in the ECM and can be released in an active formby HS degrading enzymes (33, 32, 34). It was demonstrated thatheparanase activity expressed by platelets, mast cells, neutrophils, andlymphoma cells is involved in release of active bFGF from ECM andbasement membranes (35), suggesting that heparanase activity may notonly function in cell migration and invasion, but may also elicit anindirect neovascular response. These results suggest that the ECM HSPGprovides a natural storage depot for bFGF and possibly otherheparin-binding growth promoting factors (36, 37). Displacement of bFGFfrom its storage within basement membranes and ECM may therefore providea novel mechanism for induction of neovascularization in normal andpathological situations.

[0029] Recent studies indicate that heparin and HS are involved inbinding of bFGF to high affinity cell surface receptors and in bFGF cellsignaling (38, 39). Moreover, the size of HS required for optimal effectwas similar to that of HS fragments released by heparanase (40). Similarresults were obtained with vascular endothelial cells growth factor(VEGF) (41), suggesting the operation of a dual receptor mechanisminvolving HS in cell interaction with heparin-binding growth factors. Itis therefore proposed that restriction of endothelial cell growthfactors in ECM prevents their systemic action on the vascularendothelium, thus maintaining a very low rate of endothelial cellsturnover and vessel growth. On the other hand, release of bFGF fromstorage in ECM as a complex with HS fragment, may elicit localizedendothelial cell proliferation and neovascularization in processes suchas wound healing, inflammation and tumor development (36, 37).

[0030] The Involvement of Heparanase in Other Physiological Processesand its Potential Therapeutic Applications:

[0031] Apart from its involvement in tumor cell metastasis, inflammationand autoimmunity, mammalian heparanase may be applied to modulatebioavailability of heparin-binding growth factors; cellular responses toheparin-binding growth factors (e.g., bFGF, VEGF) and cytokines (IL-8)(44, 41); cell interaction with plasma lipoproteins (49); cellularsusceptibility to certain viral and some bacterial and protozoainfections (45-47); and disintegration of amyloid plaques (48).

[0032] Viral infection: The presence of heparan sulfate on cell surfaceshave been shown to be the principal requirement for the binding ofHerpes Simplex (45) and Dengue (46) viruses to cells and for subsequentinfection of the cells. Removal of the cell surface heparan sulfate byheparanase may therefore abolish virus infection. In fact, treatment ofcells with bacterial heparitinase (degrading heparan sulfate) orheparinase (degrading heparan) reduced the binding of two related animalherpes viruses to cells and rendered the cells at least partiallyresistant to virus infection (45). There are some indications that thecell surface heparan sulfate is also involved in HIV infection (47).

[0033] Neurodegenerative diseases: Heparan sulfate proteoglycans wereidentified in the prion protein amyloid plaques of Genstmann-StrausslerSyndrome, Creutzfeldt-Jakob disease and Scrape (48). Heparanase maydisintegrate these amyloid plaques, which are also thought to play arole in the pathogenesis of Alzheimer's disease.

[0034] Restenosis and atherosclerosis: Proliferation of arterial smoothmuscle cells (SMCs) in response to endothelial injury and accumulationof cholesterol rich lipoproteins are basic events in the pathogenesis ofatherosclerosis and restenosis (50). Apart from its involvement in SMCproliferation as a low affinity receptor for heparin-binding growthfactors, HS is also involved in lipoprotein binding, retention anduptake (51). It was demonstrated that HSPG and lipoprotein lipaseparticipate in a novel catabolic pathway that may allow substantialcellular and interstitial accumulation of cholesterol rich lipoproteins(49). The latter pathway is expected to be highly atherogenic bypromoting accumulation of apoB and apoE rich lipoproteins (e.g., LDL,VLDL, chylomicrons), independent of feed back inhibition by the cellularcholesterol content. Removal of SMC HS by heparanase is thereforeexpected to inhibit both SMC proliferation and lipid accumulation andthus may halt the progression of restenosis and atherosclerosis.

[0035] Pulmonary diseases: The data obtained from the literaturesuggests a possible role for GAGs degrading enzymes, such as, but notlimited to, heparanases, connective tissue activating peptide,heparinases, hyluronidases, sulfatases and chondroitinases, in reducingthe viscosity of sinuses and airway secretions with associatedimplications on curtailing the rate of infection and inflammation. Thesputum from CF patients contains at least 3% GAGs, thus contributing toits volume and viscous properties. It was shown that heparanase reducesthe viscosity of sputum of Cystic fibrosis (CF) patients (see, U.S. Pat.No. 6,153,187). Recombinant heparanase has been shown to reduceviscosity of sputum of CF patients (see, (see, U.S. Pat. No. 6,153,187).

[0036] Heparanase and/or heparanase inhibitors may thus prove useful fortreating conditions such as wound healing, angiogenesis, restenosis,atherosclerosis, inflammation, neurodegenerative diseases and viralinfections. Mammalian heparanase can be used to neutralize plasmaheparin, as a potential replacement of protamine.

[0037] Transgenic Non-Human Models of Disease: The advantages, andvalidity of studying disease processes in non-human models has been longrecognized, and such research is, for example, a requisite stage indevelopment of all drugs and therapies for use in humans. Among thepreferred species commonly used for such studies, the mouse is clearlythe mammal most extensively classified and is often the model of choice(for an extensive review of the field see Bockamp et al, PhysiolGenomics 2002;11:115-132).

[0038] Even before the widespread application of transgenic technology,many large breeders of laboratory animals invested significant effortand expense in the establishment, using traditional breeding techniques,of mouse strains bearing phenotypes useful for the study of specificdiseases and/or treatments. Jackson Laboratories, for example(www.jax.org) offer over 300 stock strains of inbred, hybrid,wild-derived inbred and recombinant inbred mice with well-definedphenotypic characteristics.

[0039] Using genomic engineering technology, however, specificalterations in genotype, and their phenotypic effects can now be studiedwith greater precision at a fraction of the cost and time required forbreeding stock strains. For example, Jackson Laboratories today offerthousands of stock strains of transgenic mouse models for investigationin the fields of, inter alia, Cancer research, Diabetes and Obesity,Cardiovascular Disease, Immunology and Neurobiology.

[0040] Transgenic models of human disease are often produced byintroduction into mice of disease-associated transgenes bearingpreviously identified alterations of coding and regulatory sequences,for the comparison of their phenotypic effects with knowncharacteristics of the human disease. For example, the Oncomouse™(DuPont Nemours, Inc.) strains, bearing a variety of oncogene mutations,have become indispensible tools for Cancer research.

[0041] Transgenic mouse models can also be engineered to expressproteins known to be associated with human disease in conditions havingunclear etiology, providing researchers with tools to investigatedisease processes, complex interactions with multiple pathogenicfactors, combinations of risk factors and susceptibility to disease.Examples include mouse models of Alzheimer's disease expressing amyloidprotein (U.S. Pat. No. 6,509,515 to Hsiao et al) and tau filaments(Tatebayashi Y et al. PNAS USA 2002;99:13896-901); mouse models ofDiabetes Mellitus expressing human islet amyloid polypeptide (Janson etal PNAS USA 1996;93:7283-88); mouse models of colorectal cancerexpressing human carcinoembryonic antigen (CEA) (Wilkinson R W et alPNAS USA 2001;98:10256-60); mouse models of Duchenne's MuscularDystrophy overexpressing human caveolin-3 (Galbiati F et al. PNAS USA2000;97:9684-94) and mouse models of skin disease and tumorigenesisexpressing human collagenase (Darmiento, J et al Mol Cell Biol1995;15:5732-39). These transgenic mouse models also provide importanttools for evaluation of specific effects of therapies, screening ofpharmaceuticals and development of diagnostic methodologies. Thedemonstrated involvement of heparanase in immune response, inflammation,malignancy, metastasis, angiogenesis, tumorigenesis, viral infection,atherogenesis, pulmonary disease and other conditions, as detailedhereinabove, creates a strong need for a transgenic model of humanheparanse over- or under-expression.

[0042] There is, thus, a widely recognized need for, and it would behighly advantageous to have, transgenic animals producing heparanase soas to efficiently produce commercial quantities of this enzyme. Suchtransgenic animals would also find uses as models for human diseaseassociated with impaired heparanase expression, such as, for example,metastasis.

SUMMARY OF THE INVENTION

[0043] According to one aspect of the present invention there isprovided a transgenic non-human animal whose genome comprises anexogenous polynucleotide sequence integrated into the genome, theexogenous polynucleotide sequence including a promoter active in tissuesof the non-human, and a region encoding a human heparanase, wherein thepromoter and the region encoding human heparanase are operably linked inthe exogenous polynucleotide such that human heparanase is expressed inat least a portion of the cells of the non-human animal.

[0044] According to further features in the described preferredembodiments the transgenic non-human animal being homozygous orheterozygous for the exogenous polynucleotide sequence.

[0045] According to still further features in the described preferredembodiments the transgenic non-human animal having a single locus or atleast two loci each harboring the exogenous polynucleotide sequence.

[0046] According to yet further features in the described preferredembodiments the human heparanase is genetically modified to be cleavableinto an active form via a protease.

[0047] According to still further features in the described preferredembodiments the heparanase is processed by an endogenous protease of thenon-human animal into an active form.

[0048] According to yet further features in the described preferredembodiments the region of the exogenous polynucleotide sequence encodesan active form of heparanase.

[0049] According to still further features in the described preferredembodiments the transgenic non-human animal is a mammal or an avian.

[0050] According to further features in the described preferredembodiments the exogenous polynucleotide sequence includes a tissuespecific promoter for directing expression of the heparanase in a tissuespecific manner. Accordingly, the promoter is a constitutive promoterfor directing expression of the heparanase in constitutive manner or aninducible promoter for directing expression of the heparanase in aninducible manner.

[0051] According to further features in the described preferredembodiments the promoter is selected from the group consisting ofbeta-lactoglobulin promoter, Rb promoter, preproendothelin-1 promoter,beta-actin promoter, TetO promoter, metallothionein promoter, wheyacidic protein (WAP) promoter, casein promoter and lactalbumin promoter.

[0052] According to still further features in the described preferredembodiments the promoter is selected from the group consisting ofchicken lyzozyme promoter, cytomegalovirus promoter and chickenimmunoglobulin promoter.

[0053] According to yet further features in the described preferredembodiments the heparanase is expressed in, and secreted by, cells ofmammary glands of the transgenic non-human mammal.

[0054] According to still further features in the described preferredembodiments the heparanase is expressed in, and secreted by, eggproducing cells of the transgenic femal avian.

[0055] According to a further aspect of the present invention there areprovided sex cells, semen and embryos derived from the transgenicnon-human animal of the invention.

[0056] According to a further aspect of the present invention there isprovided a composition of matter comprising milk derived from anon-human transgenic mammal, the milk having detectable human heparanaseactivity.

[0057] According to a still further aspect of the present inventionthere is provided a composition of matter comprising egg yolk and/orwhite from a transgenic avian, the egg yolk and/or white havingdetectable human heparanase activity.

[0058] According to a further aspect of the present invention there isprovided a method of producing recombinant human heparanase, the methodcomprising the steps of (a) obtaining a transgenic non-human mammalhaving mammary glands, whose genome comprises an exogenouspolynucleotide sequence integrated into the genome, the exogenouspolynucleotide sequence including a promoter active in tissues of thenon-human mammal, and a region encoding a human heparanase, wherein thepromoter and the region encoding human heparanase are operably linked inthe exogenous polynucleotide such that the recombinant human heparanaseis secreted into milk being produced by the mammary glands, (b) milkingthe non-human mammal so as to obtain milk containing the recombinanthuman heparanase, and (c) purifying the recombinant human heparanasefrom the milk.

[0059] According to further features in the described preferredembodiments the promoter active in tissues of the non-human mammal is amilk protein gene promoter.

[0060] According to still further features in the described preferredembodiments the milk protein gene promoter is selected from the groupconsisting of beta-lactoglobulin promoter, Rb promoter,preproendothelin-1 promoter, whey acidic protein (WAP) promoter, caseinpromoter and lactalbumin promoter.

[0061] According to a further aspect of the present invention there isprovided a method of producing recombinant human heparanase, the methodcomprising the steps of (a) obtaining a transgenic female avian havingegg producing cells whose genome comprises an exogenous polynucleotidesequence integrated into the genome, the exogenous polynucleotidesequence including a promoter active in tissues of the transgenic femaleavian, and a region encoding a human heparanase, wherein the promoterand the region encoding human heparanase are operably linked in theexogenous polynucleotide such that the recombinant human heparanase issecreted into eggs being produced by the egg producing cells, (b)collecting eggs laid by the transgenic female avian so as to obtain eggscontaining the recombinant human heparanase and (c) purifying therecombinant human heparanase from the eggs.

[0062] According to still further features in the described preferredembodiments the promoter active in tissues of the transgenic femaleavian is an egg protein gene promoter.

[0063] According to still further features in the described preferredembodiments the egg protein gene promoter is selected from the groupconsisting of chicken lyzozyme promoter and chicken immunoglobulinpromoter.

[0064] The present invention successfully addresses the shortcomings ofthe presently known configurations by providing transgenic animalsexpressing heparanase which can be used as animal models and/or forcommercial production of recombinant heparanase.

BRIEF DESCRIPTION OF THE DRAWINGS

[0065] The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

[0066] In the drawings:

[0067]FIG. 1 presents nucleotide sequence and deduced amino acidsequence of hpa cDNA. A single nucleotide difference at position 799 (Ato T) between the EST (Expressed Sequence Tag) and the PCR amplifiedcDNA (reverse transcribed RNA) and the resulting amino acid substitution(Tyr to Phe) are indicated above and below the substituted unit,respectively. Cysteine residues and the poly adenylation consensussequence are underlined. The asterisk denotes the stop codon TGA.

[0068]FIG. 2 demonstrates degradation of soluble sulfate labeled HSPGsubstrate by lysates of High Five cells infected with pFhpa2 virus.Lysates of High Five cells that were infected with pFhpa2 virus (•) orcontrol pF2 virus (□) were incubated (18 h, 37° C.) with sulfate labeledECM-derived soluble HSPG (peak I). The incubation medium was thensubjected to gel filtration on Sepharose 6B. Low molecular weight HSdegradation fragments (peak II) were produced only during incubationwith the pFhpa2 infected cells, but there was no degradation of the HSPGsubstrate (⋄) by lysates of pF2 infected cells.

[0069]FIGS. 3a-b demonstrate degradation of soluble sulfate labeled HSPGsubstrate by the culture medium of pFhpa2 and pFhpa4 infected cells.Culture media of High Five cells infected with pFhpa2 (3 a) or pFhpa4 (3b) viruses (•), or with control viruses (□) were incubated (18 h, 37°C.) with sulfate labeled ECM-derived soluble HSPG (peak I, ⋄). Theincubation media were then subjected to gel filtration on Sepharose 6B.Low molecular weight HS degradation fragments (peak II) were producedonly during incubation with the hpa gene containing viruses. There wasno degradation of the HSPG substrate by the culture medium of cellsinfected with control viruses.

[0070]FIG. 4 presents size fractionation of heparanase activityexpressed by pFhpa2 infected cells. Culture medium of pFhpa2 infectedHigh Five cells was applied onto a 50 kDa cut-off membrane. Heparanaseactivity (conversion of the peak I substrate, (⋄) into peak II HSdegradation fragments) was found in the high (>50 kDa) (•), but not low(<50 kDa) (∘) molecular weight compartment.

[0071]FIGS. 5a-b demonstrate the effect of heparin on heparanaseactivity expressed by pFhpa2 and pFhpa4 infected High Five cells.Culture media of pFhpa2 (5 a) and pFhpa4 (5 b) infected High Five cellswere incubated (18 h, 37° C.) with sulfate labeled ECM-derived solubleHSPG (peak I, ⋄) in the absence (•) or presence (Δ) of 10 μg/ml heparin.Production of low molecular weight HS degradation fragments wascompletely abolished in the presence of heparin, a potent inhibitor ofheparanase activity (6, 7).

[0072]FIGS. 6a-b demonstrate degradation of sulfate labeled intact ECMby virus infected High Five and Sf21 cells. High Five (6 a) and Sf21 (6b) cells were plated on sulfate labeled ECM and infected (48 h, 28° C.)with pFhpa4 (•) or control pF1 (□) viruses. Control non-infected Sf21cells (R) were plated on the labeled ECM as well. The pH of the culturedmedium was adjusted to 6.0-6.2 followed by 24 h incubation at 37° C.Sulfate labeled material released into the incubation medium wasanalyzed by gel filtration on Sepharose 6B. HS degradation fragmentswere produced only by cells infected with the hpa containing virus.

[0073]FIGS. 7a-b demonstrate degradation of sulfate labeled intact ECMby virus infected cells. High Five (7 a) and Sf21 (7 b) cells wereplated on sulfate labeled ECM and infected (48 h, 28° C.) with pFhpa4(•) or control pF1 (□) viruses. Control non-infected Sf21 cells (R) wereplate on labeled ECM as well. The pH of the cultured medium was adjustedto 6.0-6.2, followed by 48 h incubation at 28° C. Sulfate labeleddegradation fragments released into the incubation medium was analyzedby gel filtration on Sepharose 6B. HS degradation fragments wereproduced only by cells infected with the hpa containing virus.

[0074]FIGS. 8a-b demonstrate degradation of sulfate labeled intact ECMby the culture medium of pFhpa4 infected cells. Culture media of HighFive (8 a) and Sf21 (8 b) cells that were infected with pFhpa4 (•) orcontrol pF1 (□) viruses were incubated (48 h, 37° C., pH 6.0) withintact sulfate labeled ECM. The ECM was also incubated with the culturemedium of control non-infected Sf21 cells (R). Sulfate labeled materialreleased into the reaction mixture was subjected to gel filtrationanalysis. Heparanase activity was detected only in the culture medium ofpFhpa4 infected cells.

[0075]FIGS. 9a-b demonstrate the effect of heparin on heparanaseactivity in the culture medium of pFhpa4 infected cells. Sulfate labeledECM was incubated (24 h, 37° C., pH 6.0) with culture medium of pFhpa4infected High Five (9 a) and Sf21 (9 b) cells in the absence (•) orpresence (V) of 10 μg/ml heparin. Sulfate labeled material released intothe incubation medium was subjected to gel filtration on Sepharose 6B.Heparanase activity (production of peak II HS degradation fragments) wascompletely inhibited in the presence of heparin.

[0076]FIGS. 10a-b demonstrate purification of recombinant heparanase onheparin-Sepharose. Culture medium of Sf21 cells infected with pFhpa4virus was subjected to heparin-Sepharose chromatography. Elution offractions was performed with 0.35-2 M NaCl gradient (⋄). Heparanaseactivity in the eluted fractions is demonstrated in FIG. 10a (•).Fractions 15-28 were subjected to 15% SDS-polyacrylamide gelelectrophoresis followed by silver nitrate staining. A correlation isdemonstrated between a major protein band (MW ˜63,000) in fractions19-24 and heparanase activity.

[0077]FIGS. 11a-b demonstrate purification of recombinant heparanase ona Superdex 75 gel filtration column. Active fractions eluted fromheparin-Sepharose (FIG. 10a) were pooled, concentrated and applied ontoSuperdex 75 FPLC column. Fractions were collected and aliquots of eachfraction were tested for heparanase activity (c, FIG. 11a) and analyzedby SDS-polyacrylamide gel electrophoresis followed by silver nitratestaining (FIG. 11b). A correlation is seen between the appearance of amajor protein band (MW ˜63,000) in fractions 4-7 and heparanaseactivity.

[0078]FIGS. 12a-e demonstrate expression of the hpa gene by RT-PCR withtotal RNA from human embryonal tissues (12 a), human extra-embryonaltissues (12 b) and cell lines from different origins (12 c-e). RT-PCRproducts using hpa specific primers (I), primers for GAPDH housekeepinggene (II), and control reactions without reverse transcriptasedemonstrating absence of genomic DNA or other contamination in RNAsamples (III). M-DNA molecular weight marker VI (Boehringer Mannheim).For 12 a: lane 1—neutrophil cells (adult), lane 2—muscle, lane 3—thymus,lane 4—heart, lane 5—adrenal. For 12 b: lane 1—kidney, lane 2—placenta(8 weeks), lane 3—placenta (11 weeks), lanes 4-7—mole (completehydatidiform mole), lane 8—cytotrophoblast cells (freshly isolated),lane 9—cytotrophoblast cells (1.5 h in vitro), lane 10—cytotrophoblastcells (6 h in vitro), lane 11—cytotrophoblast cells (18 h in vitro),lane 12—cytotrophoblast cells (48 h in vitro). For 12 c: lane 1—JARbladder cell line, lane 2—NCITT testicular tumor cell line, lane3—SW-480 human hepatoma cell line, lane 4—HTR (cytotrophoblaststransformed by SV40), lane 5—HPTLP-I hepatocellular carcinoma cell line,lane 6—EJ-28 bladder carcinoma cell line. For 12 d: lane 1—SK-hep-1human hepatoma cell line, lane 2—DAMI human megakaryocytic cell line,lane 3—DAMI cell line+PMA, lane 4—CHRF cell line+PMA, lane 5—CHRF cellline. For 12 e: lane 1—ABAE bovine aortic endothelial cells, lane 2—1063human ovarian cell line, lane 3—human breast carcinoma MDA435 cell line,lane 4—human breast carcinoma MDA231 cell line.

[0079]FIG. 13 presents a comparison between nucleotide sequences of thehuman hpa and a mouse EST cDNA fragment (SEQ ID NO:12) which is 80%homologous to the 3′ end (starting at nucleotide 1066 of SEQ ID NO:9) ofthe human hpa. The aligned termination codons are underlined.

[0080]FIG. 14 demonstrates the chromosomal localization of the hpa gene.PCR products of DNA derived from somatic cell hybrids and of genomic DNAof hamster, mouse and human of were separated on 0.7% agarose gelfollowing amplification with hpa specific primers. Lane 1—Lambda DNAdigested with BstEII, lane 2—no DNA control, lanes 3-29, PCRamplification products. Lanes 3-5—human, mouse and hamster genomic DNA,respectively. Lanes 6-29, human monochromosomal somatic cell hybridsrepresenting chromosomes 1-22 and X and Y, respectively. Lane 30—LambdaDNA digested with BstEII. An amplification product of approximately 2.8Kb is observed only in lanes 5 and 9, representing human genomic DNA andDNA derived from cell hybrid carrying human chromosome 4, respectively.These results demonstrate that the hpa gene is localized in humanchromosome 4.

[0081]FIG. 15 demonstrates the genomic exon-intron structure of thehuman hpa locus (top) and the relative positions of the lambda clonesused as sequencing templates to sequence the locus (below). The verticalrectangles represent exons (E) and the horizontal lines therebetweenrepresent introns (I), upstream (U) and downstream (D) regions.Continuous lines represent DNA fragments, which were used for sequenceanalysis. The discontinuous line in lambda 6 represent a region, whichoverlaps with lambda 8 and hence was not analyzed. The plasmid containsa PCR product, which bridges the gap between L3 and L6.

[0082]FIG. 16 presents the nucleotide sequence of the genomic region ofthe hpa gene. Exon sequences appear in upper case and intron sequencesin lower case. The deduced amino acid sequence of the exons is printedbelow the nucleotide sequence. Two predicted transcription start sitesare shown in bold.

[0083]FIG. 17 presents an alignment of the amino acid sequences of humanheparanase, mouse and partial sequences of rat homologues. The human andthe mouse sequences were determined by sequence analysis of the isolatedcDNAs. The rat sequence is derived from two different EST clones, whichrepresent two different regions (5′ and 3′) of the rat hpa cDNA. Thehuman sequence and the amino acids in the mouse and rat homologues,which are identical to the human sequence, appear in bold.

[0084]FIG. 18 presents a heparanase Zoo blot. Ten micrograms of genomicDNA from various sources were digested with EcoRI and separated on 0.7%agarose—TBE gel. Following electrophoresis, the was gel treated with HCland than with NaOH and the DNA fragments were downward transferred to anylon membrane (Hybond N+, Amersham) with 0.4 N NaOH. The membrane washybridized with a 1.6 Kb DNA probe that contained the entire hpa cDNA.Lane order: H—Human; M—Mouse; Rt—Rat; P—Pig; Cw—Cow; Hr—Horse; S—Sheep;Rb—Rabbit; D—Dog; Ch—Chicken; F—Fish. Size markers (Lambda BsteII) areshown on the left

[0085]FIG. 19 demonstrates the secondary structure prediction forheparanase performed using the PHD server—Profile network PredictionHeidelberg. H—helix, E—extended (beta strand), The glutamic acidpredicted as the proton donor is marked by asterisk and the possiblenucleophiles are underlined.

[0086] FIGS. 20A-Div demonstrate the expression of the heparanaseprotein in various tissues of homozygous transgenic mice overexpressingthe human hpa gene. 20A—Western blot analysis; 20Bi-iii—Heparanaseactivity (wild=wild type control mice; transg.=transgenic mice);20Ci-iv—Immunohistochemistry of colon and heart tissues (20Ci and20Ciii—transgenic mice, 20Cii and 20Civ—control mice). Western analysisand immunohistochemistry were performed using the anti heparanasemonoclonal antibody HP-130.

[0087] FIGS. 21A-D show morphological appearance of mammary glands(whole mount) from control (21A and 21C) vs. transgenic (21B and 21D)mice overexpressing the hpa gene in all tissues.

[0088]FIG. 22 demonstrates binding of bFGF to embryonic fibroblasts.Fibroblasts isolated from 15 days embryos of heparanase transgenic(Tg/Hep) and control mice were incubated with various concentrations of¹²⁵I-b-FGF. Following incubation cells were washed and the bound b-FGFwas quantitated.

[0089]FIG. 23 demonstrates heparanase activity in milk of transgenicmice. Milk samples from two independent lines of heparanase transgenicmice, G1 and G3, and from control mice were incubated with 35S labeledECM for 48 hours. Following incubation degradation products were sizefractionated. Heparanase activity is detected in the milk of G3 and G1transgenic mice and not in control mice.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0090] The present invention is of transgenic animals expressingheparanase, which can be used as a model for human disease and for thecommercial production of heparanase. The present invention is further ofcompositions of matter produced by the transgenic animals and of methodsof purifying heparanase therefrom. Specifically, the present inventioncan be used to produce commercial quantities of heparanase and providenon-human mammalian models of metastatic and other diseases.

[0091] The principles and operation of the present invention may bebetter understood with reference to the drawings, examples andaccompanying descriptions.

[0092] Before explaining at least one embodiment of the invention indetail, it is to be understood that the invention is not limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways. Also,it is to be understood that the phraseology and terminology employedherein is for the purpose of description and should not be regarded aslimiting.

[0093] Cloning and Expression of the Human Heparanase Gene (hpa):

[0094] The human hpa cDNA, which encodes human heparanase, was clonedfrom human placenta. It contains an open reading frame, which encodes apolypeptide of 543 amino acids with a calculated molecular weight of61,192 daltons (2). The cloning procedures of the hpa cDNA and genomicDNA from several species are described in length in U.S. Pat. No.5,986,822, U.S. patent application Ser. Nos. 09/109,386 and 09/258,892and PCT Application No. US98/17954, all of which are incorporated hereinby reference. An identical cDNA encoding human heparanase was isolatedlater on from hepatoma cell line SK-hep1 (54). From platelets (55, 57,PCT/US99/01489, PCT/AU98/00898) and from SV40 transformed fibroblasts(56, PCT/EP99/00777).

[0095] The genomic locus, which encodes heparanase, spans about 40 kb.It is composed of 12 exons separated by 11 introns and is localized onhuman chromosome 4.

[0096] The ability of the hpa gene product to catalyze degradation ofheparan sulfate (HS) in vitro was examined by expressing the entire openreading frame of hpa in High five and Sf21 insect cells, and themammalian human 293 embryonic kidney cell line expression systems.Extracts of infected or transfected cells were assayed for heparanasecatalytic activity. For this purpose, cell lysates were incubated withsulfate labeled, ECM-derived HSPG (peak I), followed by gel filtrationanalysis (Sepharose 6B) of the reaction mixture. While the substratealone consisted of high molecular weight material, incubation of theHSPG substrate with lysates of cells infected or transfected with hpacontaining vectors resulted in a complete conversion of the highmolecular weight substrate into low molecular weight labeled heparansulfate degradation fragments (see, for example, U.S. patant applicationSer. No. 09/071,618, which is incorporated herein by reference.

[0097] In other experiments, it was demonstrated that the heparanaseenzyme expressed by cells infected with a pFhpa virus is capable ofdegrading HS complexed to other macromolecular constituents (e.g.,fibronectin, laminin, collagen) present in a naturally produced intactECM (see U.S. patent application Ser. No. 09/109,386, which isincorporated herein by reference), in a manner similar to that reportedfor highly metastatic tumor cells or activated cells of the immunesystem (7, 8).

[0098] In human primary fibroblasts transfected with the heparanase cDNAthe enzyme was localized to the lysosomes.

[0099] Preferential Expression of the hpa Gene in Human Breast andHepatocellular Carcinomas:

[0100] Semi-quantitative RT-PCR was employed to evaluate the expressionof the hpa gene by human breast carcinoma cell lines exhibitingdifferent degrees of metastasis. A marked increase in hpa geneexpression is observed, which correlates to metastatic capacity ofnon-metastatic MCF-7 breast carcinoma, moderately metastatic MDA 231 andhighly metastatic MDA 435 breast carcinoma cell lines. Significantly,the differential pattern of the hpa gene expression correlated with thepattern of heparanase activity.

[0101] Expression of the hpa gene in human breast carcinoma wasdemonstrated by in situ hybridization to archival paraffin embeddedhuman breast tissue. Hybridization of the heparanase antisense riboprobeto invasive duct carcinoma tissue sections resulted in a massivepositive staining localized specifically to the carcinoma cells. The hpagene was also expressed in areas adjacent to the carcinoma showingfibrocystic changes. Normal breast tissue derived from reductionmammoplasty failed to express the hpa transcript. High expression of thehpa gene was also observed in tissue sections derived from humanhepatocellular carcinoma specimens but not in normal adult liver tissue.Furthermore, tissue specimens derived from adenocarcinoma of the ovary,squamous cell carcinoma of the cervix and colon adenocarcinoma exhibitedstrong staining with the hpa RNA probe, as compared to a very lowstaining of the hpa mRNA in the respective non-malignant control tissues(2).

[0102] A preferential expression of heparanase in human tumors versusthe corresponding normal tissues was also noted by immunohistochemicalstaining of paraffin embedded sections with monoclonal anti-heparanaseantibodies. Positive cytoplasmic staining was found in neoplastic cellsof the colon carcinoma and in dysplastic epithelial cells of atubulovillous adenoma found in the same specimen while there was littleor no staining of the normal looking colon epithelium located away fromthe carcinoma. Of particular significance was an intense immunostainingof colon adenocarcinoma cells that had metastasized into lymph node,lung and liver, as compared to the surrounding normal tissues (58).

[0103] Latent and Active Forms of the Heparanase Protein:

[0104] The apparent molecular size of the recombinant enzyme produced inthe baculovirus expression system was about 65 kDa. This heparanasepolypeptide contains 6 potential N-glycosylation sites. Followingdeglycosylation by treatment with peptide N-glycosidase, the proteinappeared as a 57 kDa band. This molecular weight corresponds to thededuced molecular mass (61,192 daltons) of the 543 amino acidpolypeptide encoded by the full length hpa cDNA after cleavage of thepredicted 3 kDa signal peptide. No further reduction in the apparentsize of the N-deglycosylated protein was observed following concurrentO-glycosidase and neuraminidase treatment. Deglycosylation had nodetectable effect on enzymatic activity.

[0105] Unlike the baculovirus enzyme, expression of the full lengthheparanase polypeptide in mammalian cells (e.g., 293 kidney cells, CHO)yielded a major protein of about 50 kDa and a minor of about 65 kDa incell lysates. Comparison of the enzymatic activity of the two forms,using a semi-quantitative gel filtration assay, revealed that the 50 kDaenzyme is at least 100-200 fold more active than the 65 kDa form. Asimilar difference was observed when the specific activity of therecombinant 65 kDa baculovirus enzyme was compared to that of the 50 kDaheparanase preparations purified from human platelets, SK-hep-1 cells,or placenta. These results suggest that the 50 kDa protein is a matureprocessed form of a latent heparanase precursor. Amino terminalsequencing of the platelet heparanase indicated that cleavage occursbetween amino acids Gln¹⁵⁷ and Lys¹⁵⁸. As indicated by the hydropathicplot of heparanase, this site is located within a hydrophillic peak,which is likely to be exposed and hence accessible to proteases.

[0106] According to Fairbank et al. (57) the precursor is cleaved atthree sites to form a heterodimer of a 50 kDa polypeptide (the matureform) that is associated with a 8 kDa peptide.

[0107] Although mammalian heparanase can be expressed in vitro in avariety of cell lines of human and non-human origin, there aresignificant drawbacks to the use of mammalian tissue culture systems forthe production of human heparanase in clinically useful quantities suchas the expense of growth media, potential contamination with host cellproteins and the limited production capacity of mammalian tissue culturesystems.

[0108] Thus, there is an important need for an efficient and relativelyinexpensive means of producing large quantities of infectiousparticle-free, human heparanase protein suitable for clinical use andresearch. The transgenic animal system described below that produceshuman heparanase recombinantly satisfies this need.

[0109] According to one aspect of the present invention there isprovided a transgenic non-human animal whose genome comprises anexogenous polynucleotide sequence integrated into the genome, theexogenous polynucleotide sequence including a promoter active in tissuesof the non-human, and a region encoding a human heparanase. The promoterand region encoding human heparanase are operably linked such that humanheparanase is expressed in at least a portion of the cells of thenon-human animal. Depending on the methods of gene transfer, and theintegration of the transgene into the host cells, the transgenicnon-human animal may be homozygous or heterozygous for the exogenouspolynucleotide sequence.

[0110] As used herein the term “animal” refers to all multicellularorganisms other than human.

[0111] As used herein, the term “transgenic” does not encompassclassical crossbreeding or in vitro fertilization, but rather denotesanimals in which one or more cells receive a recombinant DNA molecule.Although it is highly preferred that this molecule be integrated withinthe animal's chromosomes, the invention also encompasses the use ofextrachromosomally replicating DNA sequences, such as might beengineered into yeast artificial chromosomes.

[0112] As used herein the term “transgene” refers to a genetic constructincluding a polynucleotide encoding a heparanase protein. Preferably,the construct further including an additional polynucleotide harboringat least one cis-acting element which regulates the expression ofheparanase from the first polynucleotide. The cis-acting clement(s) aretypically located upstream to the coding sequence encoding heparanase.When prepared, such a construct may include additional polynucleotidesdesigned for propagating the construct in bacteria, preferably suchadditional polynucleotides are removed from the construct prior to theuse thereof for generating the transgenic animal.

[0113] The phrase “expressing heparanase from a transgene” refers totranscription of heparanase messenger RNA (mRNA) followed by translationthereof into a heparanase. Post translational modifications, includingglycosylation, proteolytic cleavage and the like may follow translation.

[0114] Heparanase catalytic activity is known to include animalendoglycosidase hydrolyzing activity which is specific for heparin orheparan sulfate proteoglycan substrates, as opposed to the activity ofbacterial enzymes (heparinase I, II and III) which degrade heparin orheparan sulfate by means of β-elimination.

[0115] Genes encoding mammalian heparanases and the expression andpurification thereof are described at length in U.S. Pat. Nos. 5,986,822and 6,177,545; U.S. patent application Ser. Nos. 09/071,618; 09/109,386;09/258,892; and PCT applications US/17954, US99/09255 and US99/09256,all of which are incorporated herein by reference. In a preferredembodiment, the gene encoding human heparanase is a polynucleotideencoding a polypeptide having heparanase catalytic activity, thepolynucleotide being at least 70%, preferably 80%, more preferably 90%and most preferably 100% homologous to nucleotide coordinates 100 to1731 of the human hpa heparanase coding sequence (GenBank Accession No.AF144325, to Vlodavsky et al), as determined using default parameters ofa DNA sequence analysis software package developed by the GeneticComputer Group (GCG) at the University of Wisconsin. In a more preferredembodiment, the gene encoding human heparanase is a polynucleotidesequence encoding a polypeptide having heparanase catalytic activity,wherein the polypeptide shares at least 70%, preferably 80% morepreferably 90% and most preferably 100% homology with human heparanase(GenBank Accession No. AAD41342 to Vlodavsky et al), as determined usingdefault parameters of a DNA sequence analysis software package developedby the Genetic Computer Group (GCG) at the University of Wisconsin.

[0116] Further details and references are provided in the Backgroundsection above. It will be appreciated by one ordinarily skilled in theart, and it is demonstrated in the above patent documents, that usingthe human heparanase gene sequence one can readily clone, express andpurify recombinant heparanase of any other mammal. This sequence ofevents, i.e., cloning a gene of one species based on the sequence of thesame gene from another species, has proven successful in hundreds ofprevious cases, especially since the polymerase chain reaction (PCR) maybe practiced therefore.

[0117] Thus, the term “heparanase” includes polypeptides encoded by amammalian heparanase gene or a portion thereof, e.g., the portionencoding the mature processed heparanase. The term also includes all ofthe heparanase species described and discussed in U.S. Pat. No.6,348,344; and in PCT/US99/09256, both are incorporated herein byreference. These species of heparanase are cleavable into active formsvia specific proteases.

[0118] The ability to incorporate specific genes into the genome ofmammalian embryos has provided a useful in vivo system for the analysisof gene control and expression. The high efficiency transformation ofcultured mammalian cells has been accomplished by direct microinjectionof specific DNA sequences into the cell nucleus (Capecchi, M., Cell 198022:479-488). Gordon, J. W. et al. (Gordon, J. W. et al. Proc. Natl.Acad. Sci. USA 1978 77:7380-7384) demonstrated that DNA could bemicroinjected into mouse embryos, and found in the resultant offspring.The basic procedure used to produce transgenic mice requires therecovery of fertilized eggs from the oviducts of newly mated femalemice. DNA, which contains the gene desired to be transferred into themouse, is microinjected into the male pronucleus of each fertilized egg.Microinjected eggs are then implanted into the oviducts of one-daypseudopregnant foster mothers and carried to term (Wagner, T. E. et al.,Proc. Natl. Acad. Sci. USA 1981 78:6376-6380). Such microinjected genesfrequently integrate into chromosomes, are retained throughoutdevelopment and are transmitted to offspring as Mendelian traits(Wagner, et al, above, and Grosschedl, R. et al. Cell October 1984;38(3):647-58). Microinjected foreign genes have shown a tendency to beexpressed in transgenic mice. Similarly, other mammalian andnon-mammalian species (e.g., avian species) are transgenized usingsimilar techniques.

[0119] Thus, a variety of transgenic animal species are presently usedto produce recombinant proteins.

[0120] For mammals, the general approach is to target the expression ofthe desired protein to the mammary gland using regulatory elementsderived from a milk protein gene and then collect and purify the productfrom milk of animals for the production of the recombinant enzyme.Transgenic cows (see, U.S. Pat. Nos. 6,080,912; 6,013,857), ewes (see,U.S. Pat. Nos. 5,756,687; 6,087,554), goats (see, U.S. Pat. No.5,843,705) and pigs (U.S. Pat. Nos. 6,030,833; 5,942,435) can be readilyengineered to produce recombinant proteins in the milk. Protocols forgenerating transgenic mammals are provided in, for example, U.S. Pat.Nos. 6,118,045; 6,018,097; 6,015,938; 5,994,616; 5,965,789; 5,965,788;5,959,171; 5,891,698; 5,880,327; 5,861,313; 5,859,307; 5,850,000;5,849,997; 5,849,992; 5,831,141; 5,827,690; 5,824,287; 5,759,536;5,756,687; 5,750,172; 5,716,817; 5,714,345; 5,705,732; 5,700,671;5,654,182; 5,648,243; 5,639,440; 5,635,355; and 5,602,300, which areincorporated herein by reference.

[0121] The following proteins have been successfully expressed in milk:lysosomal proteins; collagen, EC-SOD; bacteriostatic proteins, insulinand many more. While reducing the present invention to practice,recombinant human heparanase protein having native catalytic activitywas detected in milk of transgenic female mice expressing the humanheparanase gene hpa. Assuming an achievable expression level of 50 mg/Lin the milk of a transgenic animal of the invention and a 50% loss ofthe protein during purification, it can been estimated that about 1 cow(producing 6,000 L of milk yearly), 10 goats, sheep or pigs (producing500 L of milk yearly), or 5,333 rabbits (producing 0.9 L of milk yearly)could easily supply up to 150 grams of purified human heparanase.

[0122] Thus, according to the present invention there is provided amethod of producing recombinant human heparanase by obtaining atransgenic non-human mammal having mammary glands, whose genomecomprises an exogenous polynucleotide sequence including a promoteractive in tissues of the non-human mammal and a region encoding a humanheparanase integrated into the genome, the promoter region encodinghuman heparanase being operably linked in the exogenous polynucleotidesuch that recombinant human heparanase is secreted into milk produced bythe mammary glands, milking the non-human mammal so as to obtain milkcontaining the recombinant human heparanase, and purifying therecombinant human heparanase from the milk.

[0123] Further, according to yet another aspect of the present inventionthere is provided a composition of matter comprising milk derived from anon-human transgenic mammal, the milk having detectable human heparanaseactivity. Methods of detecting human heparanase activity include, forexample, labeled heparin degradation as described in the Materials andMethods section hereinbelow.

[0124] Obtaining milk from a transgenic animal according to the presentinvention is accomplished by conventional means. See, e.g., McBurney etal., J. Lab. Clin. Med. 64: 485 (1964); Velander et al., Proc Natl.Acad. Sci. USA 89: 12003 (1992), the respective contents of which areincorporated by reference. Heparanase or protein products thereof can beisolated and purified from milk or urine by conventional means withoutdeleteriously affecting activity. A preferred method consists of acombination of anion exchange and immunochromatographies,cryoprecipitations, zinc ion-induced precipitation of either whole milkor milk whey (defatted milk) proteins. For these techniques, see Bringeet al., J. Dairy Res. 56: 543 (1989), the contents of which areincorporated herein by reference.

[0125] Importantly, milk is known to contain a number of proteases thathave the potential to degrade foreign proteins. These include in themain an alkaline protease with tryptic and chymotryptic activities, aserine protease, a chymotrypsin-like enzyme, an aminopeptidase and anacid protease. As described hereinabove, native heparanase is cleaved byproteolytic enzymes into it's active form. Thus, in one preferredembodiment the transgenic, human heparanase is genetically modified tobe cleavable into an active form via a protease. In a most preferredembodiment, the heparanase is processed by an endogenous protease of theanimal into an active form.

[0126] Alternatively, it may be desirable to protect newly secretedheparanase against proteolytic degradation. Such precautions includerapid processing of the milk after collection and addition to the milkof well known inhibitors of proteolysis, such as are listed in SIGMACHEMICAL CO. CATALOG (1993 edition) at page 850, the contents of whichare incorporated herein by reference. Thus, in a yet further embodiment,the heparanase transgene encodes a processed and active form ofheparanase.

[0127] In addition, recombinant heparanase may be produced in eggs oftransgenic hens. The general approach in this case is to target theexpression of the desired protein to the egg-producing cells usingregulatory elements derived from an egg protein gene, and then use theegg content as a source of heparanase (e.g., collect and purify theproduct from eggs of animals for the production of the recombinantenzyme).

[0128] Methods for generating transgenic avians, and for production ofrecombinant proteins secreted into their eggs are provided, for examplein U.S. Pat. Nos. 6,080,912; 6,018,097, 5,162,255, 5,854,038. Rapp et al(U.S. patent application Publication No. 20020108132 to Rapp et al.)describe a variety of methods for introduction and expression oftrangenes in avian hosts, such as sperm-mediated transfection employingliposomes, direct microinjection of the chick embryos and nucleartransfer. Constructs for secretion of foreign proteins in chicken eggsusing chicken lyzozyme gene regulatory sequences (Lampard G R, andVerrinder Gibbins A M, Biochem Cell Biol 2002;80:777-88) andcytomegalovirus promoter (Harvey, A J et al, Nat Biotechnol2002;20:396-9) have been used successfully for stable expression anddirection of biologically active recombinant proteins to the egg whiteof transgenic chickens. Additionally, chick immunoglobulins are secretedinto yolks of developing eggs in large amounts, and their promoters andregulatory sequences can also be useful for expression and transport offoreign proteins in transgenic chicken eggs (see, for example, MorrisonS L et al 2002;38:619-625). Using the constructs described hereinabove,human heparanase can be expressed in avian eggs and purified from yolkor egg white.

[0129] Thus, according to yet another aspect of the present inventionthere is provided a method of producing heparanase by obtaining atransgenic female avian having egg producing cells whose genomecomprises an exogenous polynucleotide sequence including a promoteractive in tissues of the transgenic female avian, and a region encodinga human heparanase integrated into the genome, the promoter and regionencoding human heparanase being operably linked such that therecombinant human heparanase is secreted into eggs being produced by eggproducing cells, collecting eggs laid by the transgenic female avian soas to obtain eggs containing the human recombinant heparanase, andpurifying the recombinant human heparanase from the eggs.

[0130] Thus, according to one aspect of the present invention, there isprovided a of matter comprising egg yolk and/or white from transgenicavian, the egg yolk and/or white having detectable human heparanaseactivity.

[0131] Methods of purifying heparanase are described in, for example,U.S. Pat. No. 6,348,344 and U.S. patent application Ser. No. 09/071,618,which are incorporated herein by reference.

[0132] As is well known in the art, a transgenic animal may include asingle locus or several loci harboring the transgene. Southern blotanalysis using specific restriction endonucleases can be used to monitorthe number of copies of a transgene, so as quantitative PCR. In aspecific animal, each such loci may be homozygous or heterozygote.Careful breeding with wild type animals can be used to obtain homozygoteor heterozygote animals. In addition, a transgene can be passed from afirst genetic background of a first mating strain of a species toanother genetic background of a second mating strain of that species bycarefully implemented, and well known, breeding protocols. Typically,3-5 generations are required to do so, depending on the level ofheterogeneity between the matting strains.

[0133] The expression of the heparanase transgene may be tissuespecific, non-specific (all or most tissues), inducible or constitutive.To this end any one of a great repertoire of tissue specific,non-specific, inducible or constitutive promoters can be used. Tissuespecific promoters include, but are not limited to, beta-lactoglobulinpromoter (Genebank Accession No. X52581), mammary glands (Clark 1998) Rbpromoter (Genebank Accession No. M86180), nervous system (Jiang et al.2000), preproendothelin-1 promoter (Genebank Accession No. U07982), andcardiovascular system (Zaidi et al. 1999). Non tissue-specificconstitutive promoters include, but are not limited to, beta-actinpromoter and cytomegalovirus promoter. Inducible promoters include, butare not limited to,TetO (tet operator) promoter which is induced bydoxycycline and metallothionein promoter (Genebank Accession No.X00504). Metallothionein expression is normally low in most tissues.High expression can be induced by several inflammatory cytokines,protein kinase C activators, and stress agents including heavy metals(Mirault M E et al. Ann N Y Acad Sci Nov. 17, 1994; 738:104-15).

[0134] In addition to the abovementioned promoters, using informationderived from EST libraries, one can identify tissue specific ornon-specific mRNAs and readily clone the promoters responsible for theirexpression, which reside upstream to the coding sequence in therespective genome. Highly preferred are promoters that are specificallyactive in mammary gland cells and that involve milk proteins. Among suchpromoters, highly preferred are the short and long WAP, short and longalpha, beta and kappa casein, alpha-lactalbumin and beta-lactoglobulin(“BLG”) promoters.

[0135] Promoters may be selected on the basis of the proteincompositions of various milks. For example, the WAP and BLG promotersare particularly useful with transgenic rodents, pigs and sheep. Therodent WAP short and long promoters have been used to express the ratWAP gene, the human tPA gene and the CD4 gene, while the sheep BLGpromoter has been used to express the sheep BLG gene, the humanalpha-1-antitrypsin gene and the human Factor IX gene. For a review seeClark et al., TIBTECH 5: 20 (1987), the respective content of which isincorporated herein by reference. Preferred among the promoters forcarrying out the present invention are the rodent casein and WAPpromoters, and the casein, alpha-lactalbumin and BLG promoters fromporcine, bovine, equine and ovine (pigs, sheep, goats, cows, horses),rabbits, rodents and domestic pets (dogs and cats). The genes for thesepromoters have been isolated and their characterizations published. Forreviews see Clark et al. (1987), above, and Henninghausen, ProteinExpression and Purification4 1: 3 (1990), the respective contents ofwhich are incorporated herein by reference.

[0136] DNA sequence information is available for many mammary glandspecific genes, in at least one, and often in several organisms. See,e.g., Richards et al., J Biol. Chem. 256, 526-532 (1981)(alpha-lactalbumin rat); Campbell et al., Nucleic Acids Res. 12,8685-8697 (1984) (rat WAP); Jones et al., J Biol. Chem. 260, 7042-7050(1985) (rat beta-casein); Yu-Lee & Rosen, J Biol. Chem. 258, 10794-10804(1983) (rat gamma-casein); Hall, Biochem. J 242, 735-742 (1987)(alpha-lactalbumin human); Stewart, Nucleic Acids Res. 12, 389 (1984)(bovine alpha s1 and kappa casein cDNAs); Gorodetsky et al., Gene 66,87-96 (1988) (bovine beta casein); Alexander et al., Eur. J Biochem.178, 395-401 (1988) (bovine kappa casein); Brignon et al., FEBS Lett.188,48-55 (1977) (bovine alpha S2 casein); Jamieson et al., Gene 61,85-90 (1987), Ivanov et al., Biol. Chem. Hoppe-Seyler 369, 425-429(1988), Alexander et al., Nucleic Acids Res. 17, 6739 (1989) (bovinebeta lactoglobulin); Vilotte et al., Biochimie 69, 609-620 (1987)(bovine alpha-lactalbumin). The structure and function of the variousmilk protein genes are reviewed by Mercier & Vilotte, J Dairy Sci. 76,3079-3098 (1993) (incorporated by reference in its entirety for allpurposes). If additional flanking sequence are useful in optimizingexpression, such sequences can be cloned using the existing sequences asprobes.

[0137] Also important to the present invention are regulatory sequencesthat direct secretion of proteins into milk and/or other body fluids ofthe transgenic animal. In this regard, both homologous and heterologousregulatory sequences are useful in the invention. Mammary-gland specificregulatory sequences from different organisms can be obtained byscreening libraries from such organisms using known cognate nucleotidesequences, or antibodies to cognate proteins as probes. Generally,regulatory sequences known to direct the secretion of milk proteins,such as either signal peptides from milk or the nascent targetpolypeptide, can be used, although signal sequences can also be used inaccordance with this invention that direct the secretion of expressedproteins into other body fluids, particularly blood and urine. Mostpreferred for the transgenic mouse are the regulatory sequences for theWAP protein.

[0138] Tissue specific or constitutive expression can be used accordingto the present invention not only to produce commercial quantities ofheparanase, as described above and exemplified in the examples sectionthat follows, but also to generate animal models for a variety of humandiseases and for other applications as is further delineatedhereinafter. Methods for the generation and use of transgenic mousemodels of human disease are described in detail in, for example, U.S.Pat. Nos. 6,509,515; 6,512,161; 6,515,197; 6,521,815, and in referencesdescribed hereinabove.

[0139] Any one or more of several methods can be used to monitor theexpression of a transgene. These include tissue specific Northern blot;tissue specific RT-PCR; in situ hybridization; immunohistochemistry; andprotein activity assays. These methods are well known in the art and aredescribed in detail in, for example, “Molecular Cloning: A laboratoryManual” Sambrook et al., (1989); “Current Protocols in MolecularBiology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al.,“Current Protocols in Molecular Biology”, John Wiley and Sons,Baltimore, Md. (1989).

[0140] Transgenic mice expressing human heparanase of the presentinvention can be crossed with other mice strains with definedsusceptibility for disease (e.g., mammary cancer, Guy et al. Proc. Natl.Acad. Sci. USA 1992, 89:10578-82, prostate cancer, Tomoyuki Shirai etal. Mutation Research 2000, 412:219-226). Since heparanase expressionhas been implicated in progression of breast cancer, for example,transgenic mice expressing human heparanse could be crossed with breastcancer-related mouse models, such as Igf1 and TGFA transgenic strains(available from Jackson Labs, Maine USA), under control of an induciblepromoter, for investigation of interaction between the transgeneproducts. Efficacy of anti-cancer drugs and therapies can also be testedin such a model with greater accuracy than in existing in vitro or invivo models. Similarly, induction of inflammation and autoimmunedisorders in heparanase overexpressing mice will shed light onheparanase involvement in such conditions. The effect of heparanaseexpression on development of which involve heparan sulfate and HS boundgrowth factors can also be evaluated and may suggest possible uses fortherapy using gene therapy or the recombinant enzyme. Such conditions,which can be induced in the transgenic animals include tissue repair(e.g., wound healing, bone repair and nerve regeneration) whereheparanase is suggested to increase the availability of HS bound growthfactors and facilitate cell proliferation and migration, as well aspathological processes, which develop as a result of insufficient bloodsupply (e.g., cerebral, cardiac and diabetic ulcer ischemia), whereheparanase is suggested to induce neovascularization. Transgenic micecan also serve as a model for studying the effect of heparanase on bonemetabolism, including osteoporosis, either age related or in response toovariohysterectomy, glucocorticoid therapy and heparin therapy and onamyloidosis, such as Alzheimer disease or renal.

[0141] Constitutive overexpression of heparanase may provide essentialinformation regarding life long effects such as chronic toxicity asreflected by life span and aging, and the effect of heparanase onfertility and reproduction considering the suggested role of heparanasein embryo implantation (63).

[0142] As described in detail hereinabove, heparanase activity iscrucial for the integrity of the ECM, and has been implicated intumorigenesis, inflammation, malignancy, viral infection, tumorangiogenesis, atherogenesis and metastasis. Thus, for example,transgenic mice overexpressing heparanase provides a powerful tool forstudying the role of heparanase, metabolism of heparan sulfate and HSbound proteins in normal and pathological processes. The transgeneexpression pattern may reflect a specific mode of proteinadministration. In animals which express the transgene constitutively inall tissues, heparanase is provided chronically and systemically.

[0143] The present invention offers several advantages over existingmodels for metastasis. Transgenic mice expressing high levels of humanheparanase can be exposed to known carcinogens and cancer risk factors,and potential for metastatic development of cancerous cells observed inthese animals. Metastatic changes provide a particular advantage inscreening protocols for agents that can be used in treatment forcancerous disease such as colorectal cancer and melanoma. Furthermore,manipulation of expression of transgene expression is well known in theart (see abovementioned US patents). Organ-specific regulatorysequences, specifically promoters, can be used to target overexpressionof the human heparanase transgene to tissues of interest. Similarly,integration of the transgene into the Y chromosome can providesex-specific expression (see, for example, Neilsen et al Canc Res1992;52:3733-38).

[0144] In addition transgenic animals provide a source for primary cellsoverexpressing heparanase, such as embryonic cells, bone marrow cells,bone marrow stromal cells, spermatogonia, keratinocytes and sex cells(spermatocytes and oocytes). Such cells can be isolated using protocolsfor cell isolation and/or enrichment which are well known in the art.Based on the observation described in the Background section above thatheparanase increases cell extravasation, such cells can be transplantedfor immunotherapy, cell and gene therapy. Similarly, transgenic organscan be used for xenotransplantation, skin and embryo implantation,whereas sex cells can be used for in vitro fertilization (oocytes) andartificial insemination (spermatocytes).

[0145] Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

[0146] Reference is now made to the following examples, which togetherwith the above descriptions, illustrate the invention in a non limitingfashion.

[0147] Generally, the nomenclature used herein and the laboratoryprocedures utilized in the present invention include molecular,biochemical, microbiological and recombinant DNA techniques. Suchtechniques are thoroughly explained in the literature. See, for example,“Molecular Cloning: A laboratory Manual” Sambrook et al., (1989);“Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M.,ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”,John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guideto Molecular Cloning”, John Wiley & Sons, New York (1988); Watson etal., “Recombinant DNA”, Scientific American Books, New York; Birren etal. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Cloning and Expressing the Heparanase Gene Materials and ExperimentalMethods

[0148] Purification and characterization of heparanase from a humanhepatoma cell line and human placenta: A human hepatoma cell line(Sk-hep-1) was chosen as a source for purification of a humantumor-derived heparanase. Purification was essentially as described inU.S. Pat. No. 5,362,641 to Fuks, which is incorporated by reference asif fully set forth herein. Briefly, 500 liter, 5×10¹¹ cells were grownin suspension and the heparanase enzyme was purified about 240,000 foldby applying the following steps: (i) cation exchange (CM-Sephadex)chromatography performed at pH 6.0, 0.3-1.4 M NaCl gradient; (ii) cationexchange (CM-Sephadex) chromatography performed at pH 7.4 in thepresence of 0.1% CHAPS, 0.3-1.1 M NaCl gradient; (iii) heparin-Sepharosechromatography performed at pH 7.4 in the presence of 0.1% CHAPS,0.35-1.1 M NaCl gradient; (iv) ConA-Sepharose chromatography performedat pH 6.0 in buffer containing 0.1% CHAPS and 1 M NaCl, elution with0.25 M α-methyl mannoside; and (v) HPLC cation exchange (Mono-S)chromatography performed at pH 7.4 in the presence of 0.1% CHAPS, 0.25-1M NaCl gradient.

[0149] Active fractions were pooled, precipitated with TCA and theprecipitate subjected to SDS polyacrylamide gel electrophoresis and/ortryptic digestion and reverse phase HPLC. Tryptic peptides of thepurified protein were separated by reverse phase HPLC (C8 column) andhomogeneous peaks were subjected to amino acid sequence analysis.

[0150] The purified enzyme was applied to reverse phase HPLC andsubjected to N-terminal amino acid sequencing using the amino acidsequencer (Applied Biosystems).

[0151] Cells: Cultures of bovine corneal endothelial cells (BCECs) wereestablished from steer eyes as previously described (31, 72). Stockcultures were maintained in DMEM (1 g glucose/liter) supplemented with10% newborn calf serum and 5% FCS. bFGF (1 ng/ml) was added every otherday during the phase of active cell growth (73, 74).

[0152] Preparation of dishes coated with ECM: BCECs (second to fifthpassage) were plated into 4-well plates at an initial density of 2×10⁵cells/ml, and cultured in sulfate-free Fisher medium plus 5% dextranT-40 for 12 days. Na₂ ³⁵SO₄ (25 μCi/ml) was added on day 1 and 5 afterseeding and the cultures were incubated with the label without mediumchange. The subendothelial ECM was exposed by dissolving (5 min., roomtemperature) the cell layer with PBS containing 0.5% Triton X-100 and 20mM NH₄OH, followed by four washes with PBS. The ECM remained intact,free of cellular debris and firmly attached to the entire area of thetissue culture dish (31, 34).

[0153] To prepare soluble sulfate labeled proteoglycans (peak Imaterial), the ECM was digested with trypsin (25 μg/ml, 6 h, 37° C.),the digest was concentrated by reverse dialysis and the concentratedmaterial was applied onto a Sepharose 6B gel filtration column. Theresulting high molecular weight material (Kav<0.2, peak I) wascollected. More than 80% of the labeled material was shown to becomposed of heparan sulfate proteoglycans (26, 75).

[0154] Heparanase activity: Cells (1×10⁶/35-mm dish), cell lysates orconditioned media were incubated on top of ³⁵S-labeled ECM (18 h, 37°C.) in the presence of 20 mM phosphate buffer (pH 6.2). Cell lysates andconditioned media were also incubated with sulfate labeled peak Imaterial (10-20 μl). The incubation medium was collected, centrifuged(18,000×g, 4° C., 3 min.), and sulfate labeled material analyzed by gelfiltration on a Sepharose CL-6B column (0.9×30 cm). Fractions (0.2 ml)were eluted with PBS at a flow rate of 5 ml/h and counted forradioactivity using Bio-fluor scintillation fluid. The excluded volume(V_(o)) was marked by blue dextran and the total included volume (V_(t))by phenol red. The latter was shown to comigrate with free sulfate (76,26, 35). Degradation fragments of HS side chains were eluted fromSepharose 6B at 0.5<Kav<0.8 (peak II) (76, 26,35). A nearly intact HSPGreleased from ECM by trypsin—and, to a lower extent, during incubationwith PBS alone—was eluted next to V_(o) (Kav<0.2, peak I). Recoveries oflabeled material applied on the columns ranged from 85 to 95% indifferent experiments (26). Each experiment was performed at least threetimes and the variation of elution positions (Kav values) did not exceed+/−15%.

[0155] Cloning of hpa cDNA: cDNA clones 257548 and 260138 were obtainedfrom the I.M.A.G.E Consortium (2130 Memorial Parkway SW, Hunstville,Ala. 35801). The cDNAs were originally cloned in EcoRI and NotI cloningsites in the plasmid vector pT3T7D-Pac. Although these clones arereported to be somewhat different, DNA sequencing demonstrated thatthese clones are identical to one another. Marathon RACE (rapidamplification of cDNA ends) human placenta (poly-A) cDNA composite was agift of Prof. Yossi Shiloh of Tel Aviv University. This composite isvector free, as it includes reverse transcribed cDNA fragments to whichdouble, partially single stranded adapters are attached on both sides.The construction of the specific composite employed is described inreference 77.

[0156] Amplification of hp3 PCR fragment was performed according to theprotocol provided by Clontech laboratories. The template used foramplification was a sample taken from the above composite. The primersused for amplification were:

[0157] First step: 5′-primer: AP1: 5′-CCATCCTAATACGACTCACT ATAGGGC-3′,SEQ ID NO:1; 3′-primer: HPL229: 5′-GTAGTGATGCCA TGTAACTGAATC-3′, SEQ IDNO:2.

[0158] Second step: nested 5′-primer: AP2: 5′-ACTCACTATAGGGCTCGAGCGGC-3′, SEQ ID NO:3; nested 3′-primer: HPL171:5′-GCATCTTAGCCGTCTTTCTTCG-3′, SEQ ID NO:4. The HPL229 and HPL171 wereselected according to the sequence of the EST clones. They includenucleotides 933-956 and 876-897 of SEQ ID NO:9, respectively.

[0159] PCR program was 94° C.-4 min., followed by 30 cycles of 94° C.-40sec., 62° C.-1 min., 72° C.-2.5 min. Amplification was performed withExpand High Fidelity (Boehringer Mannheim). The resulting ca. 900 bp hp3PCR product was digested with BfrI and PvuII. Clone 257548 (phpa1) wasdigested with EcoRI, followed by end filling and was then furtherdigested with BfrI. Thereafter the PvuII-BfrI fragment of the hp3 PCRproduct was cloned into the blunt end—BfrI end of clone phpal whichresulted in having the entire cDNA cloned in pT3T7-pac vector,designated phpa2.

[0160] RT-PCR: RNA was prepared using TRI-Reagent (Molecular researchcenter Inc.) according to the manufacturer instructions. 1.25 μg weretaken for reverse transcription reaction using MuMLV Reversetranscriptase (Gibco BRL) and Oligo (dT)₁₅ primer, SEQ ID NO:5,(Promega). Amplification of the resultant first strand cDNA wasperformed with Taq polymerase (Promega). The following primers wereused:

[0161] HPU-355: 5′-TTCGATCCCAAGAAGGAATCAAC-3′, SEQ ID NO:6, nucleotides372-394 in SEQ ID NOs:9 or 11.

[0162] HPL-229: 5′-GTAGTGATGCCATGTAACTGAATC-3′, SEQ ID NO:7, nucleotides933-956 in SEQ ID NOs:9 or 11.

[0163] PCR program: 94° C.-4 min., followed by 30 cycles of 94° C.-40sec., 62° C.-1 min., 72° C.-1 min.

[0164] Alternatively, total RNA was prepared from cell cultures usingTri-reagent (Molecular Research Center, Inc.) according to themanufacturer recommendation. Poly A+ RNA was isolated from total RNAusing mRNA separator (Clontech). Reverse transcription was performedwith total RNA using Superscript II (GibcoBRL). PCR was performed withExpand high fidelity (Bochringer Mannheim). Primers used foramplification were as follows: SEQ ID NO:24 Hpu-685,5′-GAGCAGCCAGGTGAGCCCAAGAT-3′, SEQ ID NO:25 Hpu-355,5′-TTCGATCCCAAGAAGGAATCAAC-3′, SEQ ID NO:26 Hpu 565,5′-AGCTCTGTAGATGTGCTATACAC-3′, SEQ ID NO:27 Hpl 967,5′-TCAGATGCAAGCAGCAACTTTGGC-3′, SEQ ID NO:28 Hpl 171,5′-GCATCTTAGCCGTCTTTCTTCG-3′, SEQ ID NO:29 Hpl 229,5′-GTAGTGATGCCATGTAACTGAATC-3′,

[0165] PCR reaction was performed as follows: 94° C. 3 minutes, followedby 32 cycles of 94° C. 40 seconds, 64° C. 1 minute, 72° C. 3 minutes,and one cycle 72° C., 7 minutes.

[0166] Expression of recombinant heparanase in insect cells: Cells, HighFive and Sf21 insect cell lines were maintained as monolayer cultures inSF900II-SFM medium (GibcoBRL).

[0167] Recombinant Baculovirus: Recombinant virus containing the hpagene was constructed using the Bac to Bac system (GibcoBRL). Thetransfer vector pFastBac was digested with SalI and NotI and ligatedwith a 1.7 kb fragment of phpa2 digested with XhoI and NotI. Theresulting plasmid was designated pFasthpa2. An identical plasmiddesignated pFasthpa4 was prepared as a duplicate and both independentlyserved for further experimentations. Recombinant bacmid was generatedaccording to the instructions of the manufacturer with pFasthpa2,pFasthpa4 and with pFastBac. The latter served as a negative control.Recombinant bacmid DNAs were transfected into Sf21 insect cells. Fivedays after transfection recombinant viruses were harvested and used toinfect High Five insect cells, 3×10⁶ cells in T-25 flasks. Cells wereharvested 2-3 days after infection. 4×10⁶ cells were centrifuged andresuspended in a reaction buffer containing 20 mM phosphate citratebuffer, 50 mM NaCl. Cells underwent three cycles of freeze and thaw andlysates were stored at −80° C. Conditioned medium was stored at 4° C.

[0168] Partial purification of recombinant heparanase: Partialpurification of recombinant heparanase was performed byheparin-Sepharose column chromatography followed by Superdex 75 columngel filtration. Culture medium (150 ml) of Sf21 cells infected withpFhpa4 virus was subjected to heparin-Sepharose chromatography. Elutionof 1 ml fractions was performed with 0.35-2 M NaCl gradient in presenceof 0.1% CHAPS and 1 mM DTT in 10 mM sodium acetate buffer, pH 5.0. A 25μl sample of each fraction was tested for heparanase activity.Heparanase activity was eluted at the range of 0.65-1.1 M NaCl(fractions 18-26, FIG. 10a). 5 μl of each fraction was subjected to 15%SDS-polyacrylamide gel electrophoresis followed by silver nitratestaining. Active fractions eluted from heparin-Sepharose (FIG. 10a) werepooled and concentrated (×6) on YM3 cut-off membrane. 0.5 ml of theconcentrated material was applied onto 30 ml Superdex 75 FPLC columnequilibrated with 10 mM sodium acetate buffer, pH 5.0, containing 0.8 MNaCl, 1 mM DTT and 0.1% CHAPS. Fractions (0.56 ml) were collected at aflow rate of 0.75 ml/min. Aliquots of each fraction were tested forheparanase activity and were subjected to SDS-polyacrylamide gelelectrophoresis followed by silver nitrate staining (FIG. 11b).

[0169] PCR amplification of genomic DNA: 94° C. 3 minutes, followed by32 cycles of 94° C. 45 seconds, 64° C. 1 minute, 68° C. 5 minutes, andone cycle at 72° C., 7 minutes. Primers used for amplification ofgenomic DNA included:

[0170] GHpu-L3 5′-AGGCACCCTAGAGATGTTCCAG-3′, SEQ ID NO:30

[0171] GHpl-L6 5′-GAAGATTTCTGTTTCCATGACGTG-3′, SEQ ID NO:31.

[0172] Screening of genomic libraries: A human genomic library in Lambdaphage EMBLE3 SP6/T7 (Clontech, Paulo Alto, Calif.) was screened. 5×10⁵plaques were plated at 5×10⁴ pfu/plate on NZCYM agar/top agarose plates.Phages were absorbed on nylon membranes in duplicates (Qiagen).Hybridization was performed at 65° C. in 5×SSC, 5× Denhart's, 10%dextran sulfate, 100 μg/ml Salmon sperm, ³²p labeled probe (10⁶ cpm/ml).A 1.6 kb fragment, containing the entire hpa cDNA was labeled by randompriming (Boehringer Mannheim). Following hybridization membranes werewashed once with 2×SSC, 0.1% SDS at 65° C. for 20 minutes, and twicewith 0.2×SSC, 0.1% SDS at 65° C. for 15 minutes. Hybridizing plaqueswere picked, and plated at 100 pfu/plate. Hybridization was performed asabove and single isolated positive plaques were picked.

[0173] Phage DNA was extracted using a Lambda DNA extraction kit(Qiagen). DNA was digested with XhoI and EcoRI, separated on 0.7%agarose gel and transferred to nylon membrane Hybond N+ (Amersham).Hybridization and washes were performed as above.

[0174] cDNA Sequence analysis: Sequence determinations were performedwith vector specific and gene specific primers, using an automated DNAsequencer (Applied Biosystems, model 373A). Each nucleotide was readfrom at least two independent primers.

[0175] Genomic sequence analysis: Large-scale sequencing was performedby Commonwealth Biotechnology Incorporation.

[0176] Isolation of mouse hpa: Mouse hpa cDNA was amplified from eitherMarathon ready cDNA library of mouse embryo or from mRNA isolated frommouse melanoma cell line BL6, using the Marathon RACE kit from Clontech.Both procedures were performed according to the manufacturer'srecommendation.

[0177] Primers used for PCR amplification of mouse hpa: SEQ ID NO:32Mhpl773 5′-CCACACTGAATGTAATACTGAAGTG-3′, SEQ ID NO:33 MHp17365′-CGAAGCTCTGGAACTCGGCAAG-3′, SEQ ID NO:34 MHpI835′-GCCAGCTGCAAAGGTGTTGGAC-3′, SEQ ID NO:35 Mhpll525′-AACACCTGCCTCATCACGACTTC-3′, SEQ ID NO:36 Mhpll 145′-GCCAGGCTGGCGTCGATGGTGA-3′, SEQ ID NO:37 MHpI1O35′-GTCGATGGTGATGGACAGGAAC-3′, SEQ ID NO:38 - Apl 5 ′-GTAA TA CGA CTCACTA TA GGGC-3′, (Genome walker) SEQ ID NO:39 - Ap25′-ACTATAGGGCACGCGTGGT-3′, (Genome walker) SEQ ID NO:40 - Apl5′-CCATCCTAATACGACTCACTATAGGGC-3′, (Marathon RACE) SEQ ID NO:41 - Ap25′-ACTCACTATAGGGCTCGAGCGGC-3′, (Marathon RACE)

[0178] Southern analysis of genomic DNA: Genomic DNA was extracted fromanimal or from human blood using Blood and cell culture DNA maxi kit(Qiagene). DNA was digested with EcoRI, separated by gel electrophoresisand transferred to a nylon membrane Hybond N+ (Amersham). Hybridizationwas performed at 68° C. in 6×SSC, 1% SDS, 5× Denharts, 10% dextransulfate, 100 μg/ml salmon sperm DNA, and ³²p labeled probe. A 1.6 kbfragment, containing the entire hpa cDNA was used as a probe. Followinghybridization, the membrane was washed with 3×SSC, 0.1% SDS, at 68° C.and exposed to X-ray film for 3 days. Membranes were then washed with1×SSC, 0.1% SDS, at 68° C. and were reexposed for 5 days.

[0179] Construction of hpa promoter-GFP expression vector: Lambda DNA ofphage L3, was digested with SacI and BglII, resulting in a 1712 bpfragment which contained the hpa promoter (877-2688 of SEQ ID NO:42).The pEGFP-1 plasmid (Clontech) was digested with BglII and SacI andligated with the 1712 bp fragment of the hpa promoter sequence. Theresulting plasmid was designated phpEGL. A second hpa promoter-GFPplasmid was constructed containing a shorter fragment of the hpapromoter region: phpEGL was digested with HindIII, and the resulting1095 bp fragment (nucleotides 1593-2688 of SEQ ID NO:42) was ligatedwith HindIII digested pEGFP-1. The resulting plasmid was designatedphpEGS.

[0180] Computer analysis of sequences: Homology searches were performedusing several computer servers, and various databases. Blast 2.0service, at the NCBI server was used to screen the protein databaseswplus and DNA databases such as GenBank, EMBL, and the EST databases.Blast 2.0 search was performed using the basic search option of the NCBIserver. Sequence analysis and alignments were done using the DNAsequence analysis software package developed by the Genetic ComputerGroup (GCG) at the university of Wisconsin. Alignments of two sequenceswere performed using Bestfit (gap creation penalty—12, gap extensionpenalty—4). Protein homology search was performed with theSmith-Waterman algorithm, using the Bioaccelerator platform developed byCompugene. The protein database swplus was searched using the followingparameters: gapop: 10.0, gapext: 0.5, matrix: blosum62. Blocks homologywas performed using the Blocks WWW server developed at Fred HutchinsonCancer Research Center in Seattle, Wash., USA. Secondary structureprediction was performed using the PHD server—Profile network PredictionHeidelberg. Fold recognition (threading) was performed using theUCLA-DOE structure prediction server. The method used for prediction wasgonnet+predss. Alignment of three sequences was performed using thepileup application (gap creation penalty—5, gap extension penalty—1).Promoter analysis was performed using TSSW and TSSG programs (BCM SearchLauncher Human Genome Center, Baylor College of Medicine, Houston Tex.).

Example 1 Cloning of Human hpa cDNA

[0181] Purified fraction of heparanase isolated from human hepatomacells (SK-hep-1) was subjected to tryptic digestion and microsequencing.EST (Expressed Sequence Tag) databases were screened for homology to theback translated DNA sequences corresponding to the obtained peptides.Two EST sequences (accession Nos. N41349 and N45367) contained a DNAsequence encoding the peptide YGPDVGQPR (SEQ ID NO:8). These twosequences were derived from clones 257548 and 260138 (I.M.A.G.EConsortium) prepared from 8 to 9 weeks placenta cDNA library (Soares).Both clones which were found to be identical contained an insert of 1020bp which included an open reading frame (ORF) of 973 bp followed by a 3′untranslated region of 27 bp and a Poly A tail. No translation startsite (AUG) was identified at the 5′ end of these clones.

[0182] Cloning of the missing 5′ end was performed by PCR amplificationof DNA from a placenta Marathon RACE cDNA composite. A 900 bp fragment(designated hp3), partially overlapping with the identified 3′ encodingEST clones was obtained.

[0183] The joined cDNA fragment, 1721 bp long (SEQ ID NO:9), containedan open reading frame which encodes, as shown in FIG. 1 and SEQ IDNO:11, a polypeptide of 543 amino acids (SEQ ID NO:10) with a calculatedmolecular weight of 61,192 daltons. The 3′ end of the partial cDNAinserts contained in clones 257548 and 260138 started at nucleotide G⁷²¹of SEQ ID NO:9 and FIG. 1.

[0184] As further shown in FIG. 1, there was a single sequencediscrepancy between the EST clones and the PCR amplified sequence, whichled to an amino acid substitution from Tyr²⁴⁶ in the EST to Phe²⁴⁶ inthe amplified cDNA. The nucleotide sequence of the PCR amplified cDNAfragment was verified from two independent amplification products. Thenew gene was designated hpa.

[0185] As stated above, the 3′ end of the partial cDNA inserts containedin EST clones 257548 and 260138 started at nucleotide 721 of hpa (SEQ IDNO:9). The ability of the hpa cDNA to form stable secondary structures,such as stem and loop structures involving nucleotide stretches in thevicinity of position 721 was investigated using computer modeling. Itwas found that stable stem and loop structures are likely to be formedinvolving nucleotides 698-724 (SEQ ID NO:9). In addition, a high GCcontent, up to 70%, characterizes the 5′ end region of the hpa gene, ascompared to about only 40% in the 3′ region. These findings may explainthe immature termination and therefore lack of 5′ ends in the ESTclones.

[0186] To examine the ability of the hpa gene product to catalyzedegradation of heparan sulfate in an in vitro assay the entire openreading frame was expressed in insect cells, using the Baculovirusexpression system. Extracts of cells, infected with virus containing thehpa gene, demonstrated a high level of heparan sulfate degradationactivity, while cells infected with a similar construct containing nohpa gene had no such activity, nor did non-infected cells. These resultsare further demonstrated in the following Examples.

Example 2 Degradation of Soluble ECM-Derived HSPG

[0187] Monolayer cultures of High Five cells were infected (72 h, 28°C.) with recombinant Bacoluvirus containing the pFasthpa plasmid or withcontrol virus containing an insert free plasmid. The cells wereharvested and lysed in heparanase reaction buffer by three cycles offreezing and thawing. The cell lysates were then incubated (18 h, 37°C.) with sulfate labeled, ECM-derived HSPG (peak I), followed by gelfiltration analysis (Sepharose 6B) of the reaction mixture.

[0188] As shown in FIG. 2, the substrate alone included almost entirelyhigh molecular weight (Mr) material eluted next to V_(o) (peak I,fractions 5-20, Kav<0.35). A similar elution pattern was obtained whenthe HSPG substrate was incubated with lysates of cells that wereinfected with control virus. In contrast, incubation of the HSPGsubstrate with lysates of cells infected with the hpa containing virusresulted in a complete conversion of the high Mr substrate into low Mrlabeled degradation fragments (peak II, fractions 22-35, 0.5<Kav<0.75).

[0189] Fragments eluted in peak II were shown to be degradation productsof heparan sulfate, as they were (i) 5- to 6-fold smaller than intactheparan sulfate side chains (Kav approx. 0.33) released from ECM bytreatment with either alkaline borohydride or papain; and (ii) resistantto further digestion with papain or chondroitinase ABC, and susceptibleto deamination by nitrous acid (10, 26).

[0190] Similar results (not shown) were obtained with Sf21 cells. Again,heparanase activity was detected in cells infected with the hpacontaining virus (pFhpa), but not with control virus (pF). This resultwas obtained with two independently generated recombinant viruses.Lysates of control not infected High Five cells failed to degrade theHSPG substrate.

[0191] In subsequent experiments, the labeled HSPG substrate wasincubated with medium conditioned by infected High Five or Sf21 cells.

[0192] As shown in FIGS. 3a-b, heparanase activity, reflected by theconversion of the high Mr peak I substrate into the low Mr peak II whichrepresents HS degradation fragments, was found in the culture medium ofcells infected with the pFhpa2 or pFhpa4 viruses, but not with thecontrol pF1 or pF2 viruses. No heparanase activity was detected in theculture medium of control non-infected High Five or Sf21 cells.

[0193] The medium of cells infected with the pFhpa4 virus was passedthrough a 50 kDa cut off membrane to obtain a crude estimation of themolecular weight of the recombinant heparanase enzyme. As demonstratedin FIG. 4, all the enzymatic activity was retained in the uppercompartment and there was no activity in the flow through (<50 kDa)material. This result is consistent with the expected molecular weightof the hpa gene product.

[0194] In order to further characterize the hpa product the inhibitoryeffect of heparin, a potent inhibitor of heparanase mediated HSdegradation (77) was examined.

[0195] As demonstrated in FIGS. 5a-b, conversion of the peak I substrateinto peak II HS degradation fragments was completely abolished in thepresence of heparin.

[0196] Altogether, these results indicate that the heparanase enzyme isexpressed in an active form by insect cells infected with Baculoviruscontaining the newly identified human hpa gene.

Example 3 Degradation of HSPG in Intact ECM

[0197] Next, the ability of intact infected insect cells to degrade HSin intact, naturally produced ECM was investigated. For this purpose,High Five or Sf21 cells were seeded on metabolically sulfate labeled ECMfollowed by infection (48 h, 28° C.) with either the pFhpa4 or controlpF2 viruses. The pH of the medium was then adjusted to pH 6.2-6.4 andthe cells further incubated with the labeled ECM for another 48 h at 28°C. or 24 h at 37° C. Sulfate labeled material released into theincubation medium was analyzed by gel filtration on Sepharose 6B.

[0198] As shown in FIGS. 6a-b and 7 a-b, incubation of the ECM withcells infected with the control pF2 virus resulted in a constant releaseof labeled material that consisted almost entirely (>90%) of high Mrfragments (peak I) eluted with or next to V_(o). It was previously shownthat a proteolytic activity residing in the ECM itself and/or expressedby cells is responsible for release of the high Mr material (10). Thisnearly intact HSPG provides a soluble substrate for subsequentdegradation by heparanase, as also indicated by the relatively largeamount of peak I material accumulating when the heparanase enzyme isinhibited by heparin (10, 76, 78, FIG. 9). On the other hand, incubationof the labeled ECM with cells infected with the pFhpa4 virus resulted inrelease of 60-70% of the ECM-associated radioactivity in the form of lowMr sulfate-labeled fragments (peak II, 0.5<Kav<0.75), regardless ofwhether the infected cells were incubated with the ECM at 28° C. or 37°C. Control intact non-infected Sf21 or High Five cells failed to degradethe ECM HS side chains.

[0199] In subsequent experiments, as demonstrated in FIGS. 8a-b, HighFive and Sf21 cells were infected (96 h, 28° C.) with pFhpa4 or controlpF1 viruses and the culture medium incubated with sulfate-labeled ECM.Low Mr HS degradation fragments were released from the ECM only uponincubation with medium conditioned by pFhpa4 infected cells. As shown inFIG. 9, production of these fragments was abolished in the presence ofheparin. No heparanase activity was detected in the culture medium ofcontrol, non-infected cells. These results indicate that the heparanaseenzyme expressed by cells infected with the pFhpa4 virus is capable ofdegrading HS when complexed to other macromolecular constituents (i.e.fibronectin, laminin, collagen) of a naturally produced intact ECM, in amanner similar to that reported for highly metastatic tumor cells oractivated cells of the immune system (10, 76).

Example 4 Purification of Recombinant Human Heparanase

[0200] The recombinant heparanase was partially purified from medium ofpFhpa4 infected Sf21 cells by Heparin-Sepharose chromatography (FIG.10a) followed by gel filtration of the pooled active fractions over anFPLC Superdex 75 column (FIG. 11a). A ˜63 kDa protein was observed,whose quantity, as was detected by silver stained SDS-polyacrylamide gelectrophoresis, correlated with heparanase activity in the relevantcolumn fractions (FIGS. 10b and 11 b, respectively). This protein wasnot detected in the culture medium of cells infected with the controlpF1 virus and was subjected to a similar fractionation onheparin-Sepharose (not shown).

Example 5 Expression of the Human hpa cDNA in Various Cell Types, Organsand Tissues

[0201] Referring now to FIGS. 12a-e, RT-PCR was applied to evaluate theexpression of the hpa gene by various cell types and tissues. For thispurpose, total RNA was reverse transcribed and amplified. The expected585 bp long cDNA was clearly demonstrated in human kidney, placenta (8and 11 weeks) and mole tissues, as well as in freshly isolated and shorttermed (1.5-48 h) cultured human placental cytotrophoblastic cells (FIG.12a), all known to express a high heparanase activity (79). The hpatranscript was also expressed by normal human neutrophils (FIG. 12b). Incontrast, there was no detectable expression of the hpa mRNA inembryonic human muscle tissue, thymus, heart and adrenal (FIG. 12b). Thehpa gene was expressed by several, but not all, human bladder carcinomacell lines (FIG. 12c), SK hepatoma (SK-hep-1), ovarian carcinoma (OV1063), breast carcinoma (435, 231), melanoma and megakaryocytic (DAMI,CHRF) human cell lines (FIGS. 12d-e).

[0202] The above described expression pattern of the hpa transcript wasdetermined to be in a very good correlation with heparanase activitylevels determined in various tissues and cell types (not shown).

Example 6 Isolation of an Extended 5′ End of hpa cDNA from Human SK-hep1Cell Line

[0203] The 5′ end of hpa cDNA was isolated from human SK-hep1 cell lineby PCR amplification using the Marathon RACE (rapid amplification ofcDNA ends) kit (Clontech). Total RNA was prepared from SK-hep1 cellsusing the TRI-Reagent (Molecular research center Inc.) according to themanufacturer instructions. Poly A+ RNA was isolated using the mRNAseparator kit (Clonetech).

[0204] The Marahton RACE SK-hep1 cDNA composite was constructedaccording to the manufacturer recommendations. First round ofamplification was preformed using an adaptor specific primer AP1:5′-CCATCCTAATACG ACTCACTATAGGGC-3′, SEQ ID NO:1, and a hpa specificantisense primer hpl-629: 5′-CCCCAGGAGCAGCAGCATCAG-3′, SEQ ID NO:17,corresponding to nucleotides 119-99 of SEQ ID NO:9. The resulting PCRproduct was subjected to a second round of amplification using anadaptor specific nested primer AP2: 5′-ACTCACTATAGGGCTCGAGCGGC-3′, SEQID NO:3, and a hpa specific antisense nested primer hpl-6665′-AGGCTTCGAGCGCAGCAGCAT-3′, SEQ ID NO:18, corresponding to nucleotides83-63 of SEQ ID NO:9. The PCR program was as follows: a hot start of 94°C. for 1 minute, followed by 30 cycles of 90° C.-30 seconds, 68° C.-4minutes. The resulting 300 bp DNA fragment was extracted from an agarosegel and cloned into the vector pGEM-T Easy (Promega). The resultingrecombinant plasmid was designated pHPSK1.

[0205] The nucleotide sequence of the pHPSK1 insert was determined andit was found to contain 62 nucleotides of the 5′ end of the placenta hpacDNA (SEQ ID NO:9) and additional 178 nucleotides upstream, the first178 nucleotides of SEQ ID NOs:13 and 15.

[0206] A single nucleotide discrepancy was identified between theSK-hep1 cDNA and the placenta cDNA. The “T” derivative at position 9 ofthe placenta cDNA (SEQ ID NO:9), is replaced by a “C” derivative at thecorresponding position 187 of the SK-hep1 cDNA (SEQ ID NO:13).

[0207] The discrepancy is likely to be due to a mutation at the 5′ endof the placenta cDNA clone as confirmed by sequence analysis of severaladditional cDNA clones isolated from placenta, which like the SK-hep1cDNA contained C at position 9 of SEQ ID NO:9.

[0208] The 5′ extended sequence of the SK-hep1 hpa cDNA was assembledwith the sequence of the hpa cDNA isolated from human placenta (SEQ IDNO:9). The assembled sequence contained an open reading frame whichencodes, as shown in SEQ ID NOs:14 and 15, a polypeptide of 592 aminoacids with a calculated molecular weight of 66,407 daltons. The openreading frame is flanked by 93 bp 5′ untranslated region (UTR).

Example 7 Isolation of the Upstream Genomic Region of the hpa Gene

[0209] The upstream region of the hpa gene was isolated using the GenomeWalker kit (Clontech) according to the manufacturer recommendations. Thekit includes five human genomic DNA samples each digested with adifferent restriction endonuclease creating blunt ends: EcoRV, ScaI,DraI, PvuII and SspI.

[0210] The blunt ended DNA fragments are ligated to partially singlestranded adaptors. The Genomic DNA samples were subjected to PCRamplification using the adaptor specific primer and a gene specificprimer. Amplification was performed with Expand High Fidelity(Boehringer Mannheim).

[0211] A first round of amplification was performed using the ap1primer: 5′-G TAATACGACTCACTATAGGGC-3′, SEQ ID NO:19, and the hpaspecific antisense primer hpl-666: 5′-AGGCTTCGAGCGCAGCAGCAT-3′, SEQ IDNO:18, corresponding to nucleotides 83-63 of SEQ ID NO:9. The PCRprogram was as follows: a hot start of 94° C.-3 minutes, followed by 36cycles of 94° C.-40 seconds, 67° C.-4 minutes.

[0212] The PCR products of the first amplification were diluted 1:50.One μl of the diluted sample was used as a template for a secondamplification using a nested adaptor specific primer ap2:5′-ACTATAGGGCACGCGTGGT-3′, SEQ ID NO:20, and a hpa specific antisenseprimer hpl-690, 5′-CTTGGGCTCACC TGGCTGCTC-3′, SEQ ID NO:21,corresponding to nucleotides 62-42 of SEQ ID NO:9. The resultingamplification products were analyzed using agarose gel electrophoresis.Five different PCR products were obtained from the five amplificationreactions. A DNA fragment of approximately 750 bp which was obtainedfrom the SspI digested DNA sample was gel extracted. The purifiedfragment was ligated into the plasmid vector pGEM-T Easy (Promega). Theresulting recombinant plasmid was designated pGHP6905 and the nucleotidesequence of the hpa insert was determined.

[0213] A partial sequence of 594 nucleotides is shown in SEQ ID NO:16.The last nucleotide in SEQ ID NO:13 corresponds to nucleotide 93 in SEQID:13. The DNA sequence in SEQ ID NO:16 contains the 5′ region of thehpa cDNA and 501 nucleotides of the genomic upstream region which arepredicted to contain the promoter region of the hpa gene.

Example 8 Expression of the 592 Amino Acids HPA Polypeptide in a Human293 Cell Line

[0214] The 592 amino acids open reading frame (SEQ ID NOs:13 and 15) wasconstructed by ligation of the 110 bp corresponding to the 5′ end of theSK-hep1 hpa cDNA with the placenta cDNA. More specifically the MarathonRACE-PCR amplification product of the placenta hpa DNA was digested withSacI and an approximately 1 kb fragment was ligated into a SacI-digestedpGHP6905 plasmid. The resulting plasmid was digested with EarI andAatII. The EarI sticky ends were blunted and an approximately 280 bpEarI/blunt-AatII fragment was isolated. This fragment was ligated withpFasthpa digested with EcoRI which was blunt ended using Klenow fragmentand further digested with AatII. The resulting plasmid contained a 1827bp insert which includes an open reading frame of 1776 bp, 31 bp of 3′UTR and 21 bp of 5′ UTR. This plasmid was designated pFastLhpa.

[0215] A mammalian expression vector was constructed to drive theexpression of the 592 amino acids heparanase polypeptide in human cells.The hpa cDNA was excised prom pFastLhpa with BssHII and NotI. Theresulting 1850 bp BssHII-NotI fragment was ligated to a mammalianexpression vector pSI (Promega) digested with MluI and NotI. Theresulting recombinant plasmid, pSIhpaMet2 was transfected into a human293 embryonic kidney cell line.

[0216] Transient expression of the 592 amino-acids heparanase wasexamined by western blot analysis and the enzymatic activity was testedusing the gel shift assay. Both these procedures are described in lengthin U.S. Pat. No. 6,177,545, which is incorporated by reference as iffully set forth herein. Cells were harvested 3 days followingtransfection. Harvested cells were re-suspended in lysis buffercontaining 150 mM NaCl, 50 mM Tris pH 7.5, 1% Triton X-100, 1 mM PMSFand protease inhibitor cocktail (Bochringer Mannheim). 40 μg proteinextract samples were used for separation on a SDS-PAGE. Proteins weretransferred onto a PVDF Hybond-P membrane (Amersham). The membrane wasincubated with an affinity purified polyclonal anti heparanase antibody,as described in U.S. Pat. No. 6,177,545. A major band of approximately50 kDa was observed in the transfected cells as well as a minor band ofapproximately 65 kDa. A similar pattern was observed in extracts ofcells transfected with the pShpa as demonstrated in U.S. Pat. No.6,177,545. These two bands probably represent two forms of therecombinant heparanase protein produced by the transfected cells. The 65kDa protein probably represents a heparanase precursor, while the 50 kDaprotein is suggested herein to be the processed or mature form.

[0217] The catalytic activity of the recombinant protein expressed inthe pShpaMet2 transfected cells was tested by gel shift assay. Cellextracts of transfected and of mock transfected cells were incubatedovernight with heparin (6 μg in each reaction) at 37° C., in thepresence of 20 mM phosphate citrate buffer pH 5.4, 1 mM CaCl₂, 1 mM DTTand 50 mM NaCl. Reaction mixtures were then separated on a 10%polyacrylamide gel. The catalytic activity of the recombinant heparanasewas clearly demonstrated by a faster migration of the heparin moleculesincubated with the transfected cell extract as compared to the control.Faster migration indicates the disappearance of high molecular weightheparin molecules and the generation of low molecular weight degradationproducts.

Example 9 Chromosomal Localization of the hpa Gene

[0218] Chromosomal mapping of the hpa gene was performed utilizing apanel of monochromosomal human/CHO and human/mouse somatic cell hybrids,obtained from the UK HGMP Resource Center (Cambridge, England).

[0219] 40 ng of each of the somatic cell hybrid DNA samples weresubjected to PCR amplification using the hpa primers: hpu5655′-AGCTCTGTAGATGTGC TATACAC-3′, SEQ ID NO:22, corresponding tonucleotides 564-586 of SEQ ID NO:9 and an antisense primer hpl1715′-GCATCTTAGCCGTCTTTCTTCG-3′, SEQ ID NO:23, corresponding to nucleotides897-876 of SEQ ID NO:9.

[0220] The PCR program was as follows: a hot start of 94° C.-3 minutes,followed by 7 cycles of 94° C.-45 seconds, 66° C.-1 minute, 68° C.-5minutes, followed by 30 cycles of 94° C.-45 seconds, 62° C.-1 minute,68° C.-5 minutes, and a 10 minutes final extension at 72° C.

[0221] The reactions were performed with Expand long PCR (BoehringerMannheim). The resulting amplification products were analyzed usingagarose gel electrophoresis. As demonstrated in FIG. 14, a single bandof approximately 2.8 Kb was obtained from chromosome 4, as well as fromthe control human genomic DNA. A 2.8 kb amplification product isexpected based on amplification of the genomic hpa clone (data notshown). No amplification products were obtained neither in the controlDNA samples of hamster and mouse nor in somatic hybrids of other humanchromosome.

Example 10 Human Genomic Clone Encoding Heparanase

[0222] Five plaques were isolated following screening of a human genomiclibrary and were designated L3-1, L5-1, L8-1, L10-1 and L6-1. The phageDNAs were analyzed by Southern hybridization and by PCR with hpaspecific and vector specific primers. Southern analysis was performedwith three fragments of hpa cDNA: a PvuIl-BamHI fragment (nucleotides32-450, SEQ ID NO:9), a BamHI-NdeI fragment (nucleotides 451-1102, SEQID NO:9) and an NdeI-XhoI fragment (nucleotides 1103-1721, SEQ ID NO:9).

[0223] Following Southern analysis, phages L3, L6, L8 were selected forfurther analysis. A scheme of the genomic region and the relativeposition of the three phage clones is depicted in FIG. 15. A 2 kb DNAfragment containing the gap between phages L6 and L3 was PCR amplifiedfrom human genomic DNA with two gene specific primers GHpuL3 and GHpIL6.The PCR product was cloned into the plasmid vector pGEM-T-easy(Promega).

[0224] Large scale DNA sequencing of the three Lambda clones and theamplified fragment was performed with Lambda purified DNA by primerwalking. A nucleotide sequence of 44,898 bp was analyzed (FIG. 16, SEQID NO:42). Comparison of the genomic sequence with that of hpa cDNArevealed 12 exons separated by 11 introns (FIGS. 15 an 16). The genomicorganization of the hpa gene is depicted in FIG. 15 (top). The sequenceinclude the coding region from the first ATG to the stop codon whichspans 39,113 nucleotides, 2742 nucleotides upstream of the first ATG and3043 nucleotides downstream of the stop codon. Splice site consensussequences were identified at exon/intron junctions.

Example 11 Alternative Splicing

[0225] Several minor RT-PCR products were obtained from various celltypes, following amplification with hpa specific primers. Each one foundto contain a deletion of one or two exons. Some of these PCR productscontain ORFs, which encode potential shorter proteins.

[0226] Table 1 below summarizes the alternative spliced productsisolated from various cell lines.

[0227] Fragments of similar sizes were obtained following amplificationwith two cell lines, placenta and platelets. Cell type Nucleotidesdeleted Exons deleted ORF Platelets 1047-1267 8, 9 + Platelets 1154-12679 − Platelets  289-435, 562-735 2, 4 − Sk-hep1, platelets, Zr75  562-7354 + Sk-hep1 (hepatoma)  561-904 4, 5 − Zr75 (breast carcinoma)  96-203 1(partial) +

Example 12 Mouse and Rat hpa

[0228] EST databases were screened for sequences homologous to the hpagene. Three mouse EST's were identified (accession No. Aa177901, frommouse spleen, Aa067997 from mouse skin, Aa47943 from mouse embryo),assembled into a 824 bp cDNA fragment which contains a partial openreading frame (lacking a 5′ end) of 629 bp and a 3′ untranslated regionof 195 bp (SEQ ID NO:12). As shown in FIG. 13, the coding region is 80%similar to the 3′ end of the hpa cDNA sequence. These EST's are probablycDNA fragments of the mouse hpa homolog that encodes for the mouseheparanase.

[0229] Searching for consensus protein domains revealed an aminoterminal homology between the heparanase and several precursor proteinssuch as Procollagen Alpha 1 precursor, Tyrosine-protein kinase-RYK,Fibulin-1, Insulin-like growth factor binding protein and severalothers. The amino terminus is highly hydrophobic and contains apotential trans-membrane domain. The homology to known signal peptidesequences suggests that it could function as a signal peptide forprotein localization.

[0230] The amino acid sequence of human heparanase was used to searchfor homologous sequences in the DNA and protein databases. Several humanEST's were identified, as well as mouse sequences highly homologous tohuman heparanase. The following mouse EST's were identified AA177901,AA674378, AA67997, AA047943, AA690179, AI122034, all sharing anidentical sequence and correspond to amino acids 336-543 of the humanheparanase sequence. The entire mouse heparanase cDNA was cloned, basedon the nucleotide sequence of the mouse EST's. PCR primers were designedand a Marathon RACE was performed using a Marathon cDNA library from 15days mouse embryo (Clontech) and from BL6 mouse melanoma cell line. Themouse hpa homologous cDNA was isolated following several amplificationsteps. A 1.1 kb fragment was amplified from mouse embryo Marathon cDNAlibrary. The first cycle of amplification was performed with primersmhpl773 and Ap1 and the second cycle with primers mhpl736 and AP2. A 1.1kb fragment was then amplified from BL6 Marathon cDNA library. The firstcycle of amplification was performed with the primers mhpl152 and Ap1,and the second with mhpl83 and AP2. The combined sequence was homologousto nucleotides 157-1702 of the human hpa cDNA, which encode amino acids33-543. The 5′ end of the mouse hpa gene was isolated from a mousegenomic DNA library using the Genome Walker kit (Clontech). An 0.9 kbfragment was amplified from a DraI digested Genome walker DNA library.The first cycle of amplification was performed with primers mhpl114 andAp1 and the second with primers mhpl103 and AP2. The assembled sequence(SEQ ID NOs:43, 45) is 2396 nucleotides long. It contains an openreading frame of 1605 nucleotides, which encode a polypeptide of 535amino acids (SEQ ID NOs:44, 45), 196 nucleotides of 3′ untranslatedregion (UTR), and anupstream sequence which includes the promoter regionand the 5′-UTR of the mouse hpa cDNA.. According to two promoterpredicting programs TSSW and TSSG, the transcription start site islocalized to nucleotide 431 of SEQ ID NOs:43, 45, 163 nucleotidesupstream of the first ATG codon. The 431 upstream genomic sequencecontains the promoter region. A TATA box is predicted at position 394 ofSEQ ID NOs:43, 45. The mouse and the human hpa genes share an averagehomology of 78% between the nucleotide sequences and 81% similaritybetween the deduced amino acid sequences.

[0231] Search for hpa homologous sequences, using the Blast 2.0 serverrevealed two EST's from rat: AI060284 (385 nucleotides, SEQ ID NO:46)which is homologous to the amino terminus (68% similarity to amino acids12-136) of human heparanase and AI237828 (541 nucleotides, SEQ ID NO:47)which is homologous to the carboxyl terminus (81% similarity to aminoacids 500-543) of human heparanase, and contains a 3′-UTR. A comparisonbetween the human heparanase and the mouse and rat homologous sequencesis demonstrated in FIG. 17.

Example 13 Prediction of Heparanase Active Site

[0232] Homology search of heparanase amino acid sequence against the DNAand the protein databases revealed no significant homologies. Theprotein secondary structure as predicted by the PHD program consists ofalternating alpha helices and beta sheets. The fold recognition serverof UCLA predicted alpha/beta barrel structure, with under-thresholdconfidence.

[0233] Five of 15 proteins, which were predicted to have most similarfolds, were glycosyl hydrolases from various organisms: 1xyza—xylanasefrom Clostridium Thermocellum, 1pbga—6-phospho-beta-δ-galactosidase fromLactococcus Lactis, 1amy—alpha-amylase from Barley, 1ecea—endocellulasefrom Acidothermus Cellulolyticus and 1qbc—hexosaminidase alpha chain,glycosyl hydrolase.

[0234] Protein homology search using the bioaccelerator pulled outseveral proteins, including glycosyl hydrolyses such asbeta-fructofuranosidase from Vicia faba (broad bean) and from potato,lactase phlorizin hydrolase from human, xylanases from Clostridiumthermocellum and from Streptomyces halstedii and cellulase fromClostridium thermocellum. Blocks 9.3 database pulled out the active siteof glycosyl hydrolases family five, which includes cellulases fromvarious bacteria and fungi. Similar active site motif is shared byseveral lysosomal acid hydrolases (63) and other glycosyl hydrolases.The common mechanism shared by these enzymes involves two glutamic acidresidues, a proton donor and a nucleophile.

[0235] Despite the lack of an overall homology between the heparanaseand other glycosyl hydolases, the amino acid couple Asp-Glu (NE), whichis characteristic of the proton donor of glycosyl hydrolyses of the GH-Aclan, was found at positions 224-225 of the human heparanase proteinsequence. As in other clan members, this NE couple is located at the endof a β sheet.

[0236] Considering the relative location of the proton donor and thepredicted secondary structure, the glutamic acid that functions asnucleophile is most likely located at position 343, or at position 396.Identification of the active site and the amino acids directly involvedin hydrolysis opens the way for expression of the defined catalyticdomain. In addition, it will provide the tools for rational design ofenzyme activity either by modification of the microenviroment orcatalytic site itself.

Example 14 Expression of hpa Antisense in Mammalian Cell Lines

[0237] A mammalian expression vector Hpa2Kepcdna3 was constructed inorder to express hpa antisense in mammalian cells. hpa cDNA (1.7 kbEcoRI fragment) was cloned into the plasmid pCDNA3 in 3′>5′ (antisense)orientation. The construct was used to transfect MBT2-T50 and T24P celllines. 2×10⁵ cells in 35 mm plates were transfected using the Fugeneprotocol (Boehringer Mannheim). 48 hours after transfection cells weretrypsinized and seeded in six well plates. 24 hours later G418 was addedto initiate selection. The number of colonies per 35 mm plate following3 weeks: Antisense No insert T24P 15 60 MBT-T50 1 6

[0238] The lower number of colonies obtained after transfection with hpaantisense, as compared with the control plasmid suggests that theintroduction of hpa antisense interfere with cell growth. Thisexperiment demonstrates the use of complementary antisense hpa DNAsequence to control heparanase expression in cells. This approach may beused to inhibit expression of heparanase in vivo, in, for example,cancer cells and in other pathological processes in which heparanase isinvolved.

Example 15 Zoo Blot

[0239] Hpa cDNA was used as a probe to detect homologous sequences inhuman DNA and in DNA of various animals. The autoradiogram of theSouthern analysis is presented in FIG. 18. Several bands were detectedin human DNA, which correlated with the accepted pattern according tothe genomic hpa sequence. Several intense bands were detected in allmammals, while faint bands were detected in chicken. This correlateswith the phylogenetic relation between human and the tested animals. Theintense bands indicate that hpa is conserved among mammals as well as inmore genetically distant organisms. The multiple bands patterns suggestthat in all animals, like in human, the hpa locus occupy large genomicregion. Alternatively, the various bands could represent homologoussequences and suggest the existence of a gene family, which can beisolated based on their homology to the human hpa reported herein. Thisconservation was actually found, between the isolated human hpa cDNA andthe mouse homologue.

Example 16 Characterization of the hpa Promoter

[0240] The DNA sequence upstream of the hpa first ATG was subjected tocomputational analysis in order to localize the predicted transcriptionstart site and to identify potential transcription factors bindingsites. Recognition of human PolII promoter region and start oftranscription were predicted using the TSSW and TSSG programs. Bothprograms identified a promoter region upstream of the coding region.TSSW pointed at nucleotide 2644 and TSSG at 2635 of SEQ ID NO:42. Thesetwo predicted transcription start sites are located 4 and 13 nucleotidesupstream of the longest hpa cDNA isolated by RACE.

[0241] A hpa promoter-GFP reporter vector was constructed in order toinvestigate the regulation of hpa transcription. Two constructs weremade, containing 1.8 kb and 1.1 kb of the hpa promoter region. Thereporter vector was transfected into T50-mouse bladder carcinoma cells.Cells transfected with both constructs exhibited green fluorescence,which indicated the promoter activity of the genomic sequence upstreamof the hpa-coding region. This reporter vector, enables the monitoringof hpa promoter activity, at various conditions and in different celltypes and to characterize the factors involved regulation of hpaexpression.

Example 17 Human Heparanase Expressing Transgenic Mice

[0242] Materials, Methods and Experimetal Results

[0243] Immunohistochemistry:

[0244] Micrometer sections were deparaffinized and rehydrated. Tissuewas then denatured for 3 minutes in a microwave oven in citrate buffer(0.01 M, pH 6.0). Blocking steps included successive incubations in 0.2%glycine, 3% H₂O₂ in methanol and 5% goat serum. Sections were incubatedwith a monoclonal anti-human heparanase antibody HP-130 (see U.S. Pat.No. 6,177,545) diluted in PBS, or with DMEM supplemented with 10% horseserum as control, diluted as above, followed by incubation with HRPconjugated goat anti mouse IgG+IgM antibody (Jackson). Color wasdeveloped using Zymed AEC substrate kit (Zymed) for 10 minutes, followedby counter stain with Mayer's hematoxylin.

[0245] Preparation of Dishes Coated with ECM:

[0246] Bovine corneal EC were cultured as described in U.S. Pat. No.5,986,822 except that 5% dextran T-40 was included in the growth mediumand the cells were maintained without addition of bFGF for 12 days. Thesubendothelial ECM was exposed by dissolving the cell layer with PBScontaining 0.5% Triton X-100 and 20 mM NH40H, followed by four washed inPBS. The ECM remained intact, free of cellular debris and firmlyattached to the entire area of the tissue culture dish. For preparationof sulfate-labeled ECM, corneal endothelial cells were cultured in thepresence of Na₂[³⁵S]O₄ (Amersham) added (25 μCi/ml) one day and 5 daysafter seeding and the cultures were incubated with the label withoutmedium change. Ten to twelve days after seeding, the cell monolayer wasdissolved and the ECM exposed.

[0247] Heparanase Activity:

[0248] Degradation of sulfate labeled ECM by heparanase was determinedas described in U.S. Pat. No. 5,986,822. Briefly, ECM was incubated (24hours, 37° C., pH 6.2) with recombinant heparanase or hpa-transfectedcells and sulfate labeled material released into the incubation mediumwas analyzed by gel filtration on a Sepharose 6B column. Intact HSPGswere eluted just after the void volume (Kav<0.2, peak I) and HSdegradation fragments eluted with 0.5<Kav<0.8 (peak II).

[0249] Generation of Heparanase Transgenic Mice:

[0250] Human hpa cDNA was cloned from a human placenta cDNA library (seeU.S. Pat. No. 5,968,822) using back-translated DNA sequencescorresponding to peptides from human hepatoma haparanase. After fillingin missing 5′ ends in the placenta EST clones a cDNA fragment, 1721 bplong (GeneBank Accession No. AF144325), contained an open reading framewhich encodes a polypeptide of 543 amino acids (GenBank Accession No.AAD41342) with a calculated molecular weight of 61,192 daltons wasobtained. High-level constitutive expression of heparanase was driven bychicken beta-actin promoter. The plasmid pCAGGS (64) was modified tocontain a unique EcoRI site at position 1719. An XbaI-EcoRI 1.7 kbfragment, which contained the entire open reading frame of heparanasewas cloned into the compatible sites of the vector.

[0251] Before injection, the plasmid pCAGGS-hpa was digested with SalIand PstI in order to isolate the expression cassette and eliminatebacterial DNA sequences. The resulting fragment contained the CMV-IEenhancer, chicken β-actin promoter and hpa cDNA followed by a rabbitb-globin poly adenylation site.

[0252] The DNA fragment containing the hpa expression cassette wasinjected into fertilized eggs, derived from C57BL×BalbC breed. Theisolation of fertilized eggs, injection of DNA and transplantation ofblastocytes were conducted by the Department of cell biochemistry—thetransgenic unit at the Hadassah Medical School, Jerusalem according to aprotocol adapted from Hogan et al. Manipulating the Mouse Embryo ALaboratory Manual, Cold Spring Harbor Laboratory Press, 1994.

[0253] Mice developed from the injected blastocytes were tested for thepresence of the human hpa transgene in their genome. Genomic DNA wasextracted from tail tips of the mice and the human hpa transgenesequence was amplified using human hpa specific PCR primers. To thisend, tail fragments were incubated overnight at 55° C. in a lysis buffer(8 M urea, 0.2 M Tris-HCl, 0.4 M NaCl, 20 mM EDTA, 1% N-Laurylsarcosine,10 μg/ml proteinase K). The dissolved tissue underwent phenol extractionand ethanol precipitation, to obtain a highly purified genomic DNA.

[0254] The integration of the human heparanase cDNA in the mouse genomewas verified by PCR using two sets of primers. The first couple wasdesigned to amplify the 5′ region of the transgene. It included aβ-actin promoter specific primer (designated 5′-pCAGGs)5′-ATAGGCAGCTGACCTGA-3′ (SEQ ID NO:48) and human hpa specific primer:(designated Hpl-300) 5′-TGACTTGAGATTGCCAGTAACTTC-3′ (SEQ ID NO:49). Thesecond primers set was designed to amplify the 3′ region of thetransgene. It included a human hpa specific primer (designated Hpu-830)5′-CTGTCCAACTCAATGGTCTAACTC-3′ (SEQ ID NO:50), and a primer specific tothe plasmid derived 3′-untranslated region (designated 3′ pCAGGS)5′-TCTAGAGCCTCTGCTAACCA-3′ (SEQ ID NO:51); PCR conditions were asfollows: 2 minutes at 95° C. followed by 33 cycles of 15 seconds at 95°C., 1 minute at 58° C. and 1 minute at 72° C.

[0255] Four G₀ founder mice were obtained, harboring the human hpa cDNAin their genome as revealed by a PCR reaction specific for the human hpacDNA. Founders were mated with C57B1 mice to create F1 mice and thosewere mated among themselves to create F2 mice. Homozygous F2 mice fromeach G₀ line were identified by Southern blot analysis and aquantitative PCR assay. Homozygosity was verified by mating with C57B1mice, where all the pups were positive heterozygous. All foundertransgenic mice were back crossed with C57BL mice in order to establishC57B1 transgenic mice with a pure genetic background.

[0256] Expression of Human Heparanase in Transgenic Mice:

[0257] Expression of the heparanase protein was demonstrated by Westernblot analysis of tissue extracts derived from F1 transgenic and controlmice (FIG. 20A). Measurements of heparanase activity in tissue extractsrevealed a much higher activity in the transgenic as compared to controlmice in all tissues examined (FIGS. 20Bi-iii). Immunohistochemicalstaining of tissue sections revealed a strong expression of the humanheparanase protein in tissues derived from the transgenic mice, but notcontrol mice (FIGS. 20Ci-iv).

[0258] Phenotype of Human Heparanase Overexpressing Transgenic Mice:

[0259] The transgenic mice are fertile and show no apparent signs ofabnormality. Few phenotypic alterations were however noted. For example,the virgin transgenic mice develop lobular-alveoli structures in themammary gland, a phenomenon that is characteristic of mammary glands ofpregnant mice (FIGS. 21A-D).

[0260] Overexpression of heparanase may lead to alterations in theamount and composition of heparan sulfate in the extracellular matrix(ECM) and surface of cells derived from the transgenic vs. control mice.In order to examine the effect of heparanase overexpression on cellsurface heparan sulfate, the bFGF binding capacity of embryonic cellsfrom transgenic and control mice was tested. Fibroblasts were isolatedfrom embryos of transgenic mice and control mice 15 days post gestation.Cells were cultured in DMEM/RPMI/F-12 medium supplemented with 10% FCS.Confluent cells were incubated with various concentrations ofradio-iodinated bFGF. Following incubation cells were washed and thebound bFGF was quantitated. As shown in FIG. 22, binding of bFGF tofibroblasts of transgenic embryos was lower than to fibroblasts ofcontrol embryos. This observation suggests that high levels ofheparanase reduce the amount of heparan sulfate on the cell surface.

[0261] Heparanase in Milk of Transgenic Mice:

[0262] Milk of transgenic mice was tested for heparanase activity. Milkwas obtained from females of two independent lines of transgenic miceand from control mice 7-10 days after delivery. Milk was diluted 1:10 inphosphate citrate buffer pH 6.0 and incubated on 35S labeled ECM for 48hours. Degradation products were size fractionated. As shown in FIG. 23heparanase activity was detected in the two transgenic lines G1 and G3,while no activity was detected in milk of control mice. This observationindicates that active heparanase can by produced in the mammary glandsand secreted into the milk of transgenic animals.

[0263] Tissue Specific Expression of Heparanase in Transgenic Mice:

[0264] In more recent experiments, the hpa cDNA was cloned into a PES7plasmid, a derivative of pSP72 containing the minimal apoA1 promoter,driving expression of the human 7 alpha-hydroxylase enzyme exclusivelyin the liver of male mice. (PES7 expression vector was a gift fromSchayek E., Bresbow L. B, The Rockefeller University NY. The 7alpha-hydroxylase was replaced by the hpa cDNA in the properorientation. Briefly, hpa cDNA was excised from pCAGGS-hpa2 using XbaI.The 1.7 kb XbaI fragment was subcloned into the XbaI site of PES7plasmid. The appropriate linear fragment was cut, purified and subjectedto microinjection. A single transgenic mouse expressing the human hpacDNA was obtained. This mouse was bred to produce F1 mice.

[0265] It is appreciated that certain features of the invention, whichare, for clarity, described in the context of separate embodiments, mayalso be provided in combination in a single embodiment. Conversely,various features of the invention, which are, for brevity, described inthe context of a single embodiment, may also be provided separately orin any suitable subcombination.

[0266] Although the invention has been described in conjunction withspecific embodiments thereof, it is evident that many alternatives,modifications and variations will be apparent to those skilled in theart. Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents, patent applicationsand sequences identified by a Genbank accession number mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent, patent application or sequence was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention.

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1 51 1 27 DNA Artificial sequence Single strand DNA oligonucleotide 1ccatcctaat acgactcact atagggc 27 2 24 DNA Artificial sequence Singlestrand DNA oligonucleotide 2 gtagtgatgc catgtaactg aatc 24 3 23 DNAArtificial sequence Single strand DNA oligonucleotide 3 actcactatagggctcgagc ggc 23 4 22 DNA Artificial sequence Single strand DNAoligonucleotide 4 gcatcttagc cgtctttctt cg 22 5 15 DNA Artificialsequence Single strand DNA oligonucleotide 5 tttttttttt ttttt 15 6 23DNA Artificial sequence Single strand DNA oligonucleotide 6 ttcgatcccaagaaggaatc aac 23 7 24 DNA Artificial sequence Single strand DNAoligonucleotide 7 gtagtgatgc catgtaactg aatc 24 8 9 PRT Artificialsequence Peptide derived from tryptic digestion of human heparenase 8Tyr Gly Pro Asp Val Gly Gln Pro Arg 1 5 9 1721 DNA Homo sapiens 9ctagagcttt cgactctccg ctgcgcggca gctggcgggg ggagcagcca ggtgagccca 60agatgctgct gcgctcgaag cctgcgctgc cgccgccgct gatgctgctg ctcctggggc 120cgctgggtcc cctctcccct ggcgccctgc cccgacctgc gcaagcacag gacgtcgtgg 180acctggactt cttcacccag gagccgctgc acctggtgag cccctcgttc ctgtccgtca 240ccattgacgc caacctggcc acggacccgc ggttcctcat cctcctgggt tctccaaagc 300ttcgtacctt ggccagaggc ttgtctcctg cgtacctgag gtttggtggc accaagacag 360acttcctaat tttcgatccc aagaaggaat caacctttga agagagaagt tactggcaat 420ctcaagtcaa ccaggatatt tgcaaatatg gatccatccc tcctgatgtg gaggagaagt 480tacggttgga atggccctac caggagcaat tgctactccg agaacactac cagaaaaagt 540tcaagaacag cacctactca agaagctctg tagatgtgct atacactttt gcaaactgct 600caggactgga cttgatcttt ggcctaaatg cgttattaag aacagcagat ttgcagtgga 660acagttctaa tgctcagttg ctcctggact actgctcttc caaggggtat aacatttctt 720gggaactagg caatgaacct aacagtttcc ttaagaaggc tgatattttc atcaatgggt 780cgcagttagg agaagattat attcaattgc ataaacttct aagaaagtcc accttcaaaa 840atgcaaaact ctatggtcct gatgttggtc agcctcgaag aaagacggct aagatgctga 900agagcttcct gaaggctggt ggagaagtga ttgattcagt tacatggcat cactactatt 960tgaatggacg gactgctacc agggaagatt ttctaaaccc tgatgtattg gacattttta 1020tttcatctgt gcaaaaagtt ttccaggtgg ttgagagcac caggcctggc aagaaggtct 1080ggttaggaga aacaagctct gcatatggag gcggagcgcc cttgctatcc gacacctttg 1140cagctggctt tatgtggctg gataaattgg gcctgtcagc ccgaatggga atagaagtgg 1200tgatgaggca agtattcttt ggagcaggaa actaccattt agtggatgaa aacttcgatc 1260ctttacctga ttattggcta tctcttctgt tcaagaaatt ggtgggcacc aaggtgttaa 1320tggcaagcgt gcaaggttca aagagaagga agcttcgagt ataccttcat tgcacaaaca 1380ctgacaatcc aaggtataaa gaaggagatt taactctgta tgccataaac ctccataacg 1440tcaccaagta cttgcggtta ccctatcctt tttctaacaa gcaagtggat aaataccttc 1500taagaccttt gggacctcat ggattacttt ccaaatctgt ccaactcaat ggtctaactc 1560taaagatggt ggatgatcaa accttgccac ctttaatgga aaaacctctc cggccaggaa 1620gttcactggg cttgccagct ttctcatata gtttttttgt gataagaaat gccaaagttg 1680ctgcttgcat ctgaaaataa aatatactag tcctgacact g 1721 10 543 PRT Homosapiens 10 Met Leu Leu Arg Ser Lys Pro Ala Leu Pro Pro Pro Leu Met LeuLeu 1 5 10 15 Leu Leu Gly Pro Leu Gly Pro Leu Ser Pro Gly Ala Leu ProArg Pro 20 25 30 Ala Gln Ala Gln Asp Val Val Asp Leu Asp Phe Phe Thr GlnGlu Pro 35 40 45 Leu His Leu Val Ser Pro Ser Phe Leu Ser Val Thr Ile AspAla Asn 50 55 60 Leu Ala Thr Asp Pro Arg Phe Leu Ile Leu Leu Gly Ser ProLys Leu 65 70 75 80 Arg Thr Leu Ala Arg Gly Leu Ser Pro Ala Tyr Leu ArgPhe Gly Gly 85 90 95 Thr Lys Thr Asp Phe Leu Ile Phe Asp Pro Lys Lys GluSer Thr Phe 100 105 110 Glu Glu Arg Ser Tyr Trp Gln Ser Gln Val Asn GlnAsp Ile Cys Lys 115 120 125 Tyr Gly Ser Ile Pro Pro Asp Val Glu Glu LysLeu Arg Leu Glu Trp 130 135 140 Pro Tyr Gln Glu Gln Leu Leu Leu Arg GluHis Tyr Gln Lys Lys Phe 145 150 155 160 Lys Asn Ser Thr Tyr Ser Arg SerSer Val Asp Val Leu Tyr Thr Phe 165 170 175 Ala Asn Cys Ser Gly Leu AspLeu Ile Phe Gly Leu Asn Ala Leu Leu 180 185 190 Arg Thr Ala Asp Leu GlnTrp Asn Ser Ser Asn Ala Gln Leu Leu Leu 195 200 205 Asp Tyr Cys Ser SerLys Gly Tyr Asn Ile Ser Trp Glu Leu Gly Asn 210 215 220 Glu Pro Asn SerPhe Leu Lys Lys Ala Asp Ile Phe Ile Asn Gly Ser 225 230 235 240 Gln LeuGly Glu Asp Tyr Ile Gln Leu His Lys Leu Leu Arg Lys Ser 245 250 255 ThrPhe Lys Asn Ala Lys Leu Tyr Gly Pro Asp Val Gly Gln Pro Arg 260 265 270Arg Lys Thr Ala Lys Met Leu Lys Ser Phe Leu Lys Ala Gly Gly Glu 275 280285 Val Ile Asp Ser Val Thr Trp His His Tyr Tyr Leu Asn Gly Arg Thr 290295 300 Ala Thr Arg Glu Asp Phe Leu Asn Pro Asp Val Leu Asp Ile Phe Ile305 310 315 320 Ser Ser Val Gln Lys Val Phe Gln Val Val Glu Ser Thr ArgPro Gly 325 330 335 Lys Lys Val Trp Leu Gly Glu Thr Ser Ser Ala Tyr GlyGly Gly Ala 340 345 350 Pro Leu Leu Ser Asp Thr Phe Ala Ala Gly Phe MetTrp Leu Asp Lys 355 360 365 Leu Gly Leu Ser Ala Arg Met Gly Ile Glu ValVal Met Arg Gln Val 370 375 380 Phe Phe Gly Ala Gly Asn Tyr His Leu ValAsp Glu Asn Phe Asp Pro 385 390 395 400 Leu Pro Asp Tyr Trp Leu Ser LeuLeu Phe Lys Lys Leu Val Gly Thr 405 410 415 Lys Val Leu Met Ala Ser ValGln Gly Ser Lys Arg Arg Lys Leu Arg 420 425 430 Val Tyr Leu His Cys ThrAsn Thr Asp Asn Pro Arg Tyr Lys Glu Gly 435 440 445 Asp Leu Thr Leu TyrAla Ile Asn Leu His Asn Val Thr Lys Tyr Leu 450 455 460 Arg Leu Pro TyrPro Phe Ser Asn Lys Gln Val Asp Lys Tyr Leu Leu 465 470 475 480 Arg ProLeu Gly Pro His Gly Leu Leu Ser Lys Ser Val Gln Leu Asn 485 490 495 GlyLeu Thr Leu Lys Met Val Asp Asp Gln Thr Leu Pro Pro Leu Met 500 505 510Glu Lys Pro Leu Arg Pro Gly Ser Ser Leu Gly Leu Pro Ala Phe Ser 515 520525 Tyr Ser Phe Phe Val Ile Arg Asn Ala Lys Val Ala Ala Cys Ile 530 535540 11 1721 DNA Homo sapiens CDS (63)..(1691) 11 ctagagcttt cgactctccgctgcgcggca gctggcgggg ggagcagcca ggtgagccca 60 ag atg ctg ctg cgc tcgaag cct gcg ctg ccg ccg ccg ctg atg ctg 107 Met Leu Leu Arg Ser Lys ProAla Leu Pro Pro Pro Leu Met Leu 1 5 10 15 ctg ctc ctg ggg ccg ctg ggtccc ctc tcc cct ggc gcc ctg ccc cga 155 Leu Leu Leu Gly Pro Leu Gly ProLeu Ser Pro Gly Ala Leu Pro Arg 20 25 30 cct gcg caa gca cag gac gtc gtggac ctg gac ttc ttc acc cag gag 203 Pro Ala Gln Ala Gln Asp Val Val AspLeu Asp Phe Phe Thr Gln Glu 35 40 45 ccg ctg cac ctg gtg agc ccc tcg ttcctg tcc gtc acc att gac gcc 251 Pro Leu His Leu Val Ser Pro Ser Phe LeuSer Val Thr Ile Asp Ala 50 55 60 aac ctg gcc acg gac ccg cgg ttc ctc atcctc ctg ggt tct cca aag 299 Asn Leu Ala Thr Asp Pro Arg Phe Leu Ile LeuLeu Gly Ser Pro Lys 65 70 75 ctt cgt acc ttg gcc aga ggc ttg tct cct gcgtac ctg agg ttt ggt 347 Leu Arg Thr Leu Ala Arg Gly Leu Ser Pro Ala TyrLeu Arg Phe Gly 80 85 90 95 ggc acc aag aca gac ttc cta att ttc gat cccaag aag gaa tca acc 395 Gly Thr Lys Thr Asp Phe Leu Ile Phe Asp Pro LysLys Glu Ser Thr 100 105 110 ttt gaa gag aga agt tac tgg caa tct caa gtcaac cag gat att tgc 443 Phe Glu Glu Arg Ser Tyr Trp Gln Ser Gln Val AsnGln Asp Ile Cys 115 120 125 aaa tat gga tcc atc cct cct gat gtg gag gagaag tta cgg ttg gaa 491 Lys Tyr Gly Ser Ile Pro Pro Asp Val Glu Glu LysLeu Arg Leu Glu 130 135 140 tgg ccc tac cag gag caa ttg cta ctc cga gaacac tac cag aaa aag 539 Trp Pro Tyr Gln Glu Gln Leu Leu Leu Arg Glu HisTyr Gln Lys Lys 145 150 155 ttc aag aac agc acc tac tca aga agc tct gtagat gtg cta tac act 587 Phe Lys Asn Ser Thr Tyr Ser Arg Ser Ser Val AspVal Leu Tyr Thr 160 165 170 175 ttt gca aac tgc tca gga ctg gac ttg atcttt ggc cta aat gcg tta 635 Phe Ala Asn Cys Ser Gly Leu Asp Leu Ile PheGly Leu Asn Ala Leu 180 185 190 tta aga aca gca gat ttg cag tgg aac agttct aat gct cag ttg ctc 683 Leu Arg Thr Ala Asp Leu Gln Trp Asn Ser SerAsn Ala Gln Leu Leu 195 200 205 ctg gac tac tgc tct tcc aag ggg tat aacatt tct tgg gaa cta ggc 731 Leu Asp Tyr Cys Ser Ser Lys Gly Tyr Asn IleSer Trp Glu Leu Gly 210 215 220 aat gaa cct aac agt ttc ctt aag aag gctgat att ttc atc aat ggg 779 Asn Glu Pro Asn Ser Phe Leu Lys Lys Ala AspIle Phe Ile Asn Gly 225 230 235 tcg cag tta gga gaa gat tat att caa ttgcat aaa ctt cta aga aag 827 Ser Gln Leu Gly Glu Asp Tyr Ile Gln Leu HisLys Leu Leu Arg Lys 240 245 250 255 tcc acc ttc aaa aat gca aaa ctc tatggt cct gat gtt ggt cag cct 875 Ser Thr Phe Lys Asn Ala Lys Leu Tyr GlyPro Asp Val Gly Gln Pro 260 265 270 cga aga aag acg gct aag atg ctg aagagc ttc ctg aag gct ggt gga 923 Arg Arg Lys Thr Ala Lys Met Leu Lys SerPhe Leu Lys Ala Gly Gly 275 280 285 gaa gtg att gat tca gtt aca tgg catcac tac tat ttg aat gga cgg 971 Glu Val Ile Asp Ser Val Thr Trp His HisTyr Tyr Leu Asn Gly Arg 290 295 300 act gct acc agg gaa gat ttt cta aaccct gat gta ttg gac att ttt 1019 Thr Ala Thr Arg Glu Asp Phe Leu Asn ProAsp Val Leu Asp Ile Phe 305 310 315 att tca tct gtg caa aaa gtt ttc caggtg gtt gag agc acc agg cct 1067 Ile Ser Ser Val Gln Lys Val Phe Gln ValVal Glu Ser Thr Arg Pro 320 325 330 335 ggc aag aag gtc tgg tta gga gaaaca agc tct gca tat gga ggc gga 1115 Gly Lys Lys Val Trp Leu Gly Glu ThrSer Ser Ala Tyr Gly Gly Gly 340 345 350 gcg ccc ttg cta tcc gac acc tttgca gct ggc ttt atg tgg ctg gat 1163 Ala Pro Leu Leu Ser Asp Thr Phe AlaAla Gly Phe Met Trp Leu Asp 355 360 365 aaa ttg ggc ctg tca gcc cga atggga ata gaa gtg gtg atg agg caa 1211 Lys Leu Gly Leu Ser Ala Arg Met GlyIle Glu Val Val Met Arg Gln 370 375 380 gta ttc ttt gga gca gga aac taccat tta gtg gat gaa aac ttc gat 1259 Val Phe Phe Gly Ala Gly Asn Tyr HisLeu Val Asp Glu Asn Phe Asp 385 390 395 cct tta cct gat tat tgg cta tctctt ctg ttc aag aaa ttg gtg ggc 1307 Pro Leu Pro Asp Tyr Trp Leu Ser LeuLeu Phe Lys Lys Leu Val Gly 400 405 410 415 acc aag gtg tta atg gca agcgtg caa ggt tca aag aga agg aag ctt 1355 Thr Lys Val Leu Met Ala Ser ValGln Gly Ser Lys Arg Arg Lys Leu 420 425 430 cga gta tac ctt cat tgc acaaac act gac aat cca agg tat aaa gaa 1403 Arg Val Tyr Leu His Cys Thr AsnThr Asp Asn Pro Arg Tyr Lys Glu 435 440 445 gga gat tta act ctg tat gccata aac ctc cat aac gtc acc aag tac 1451 Gly Asp Leu Thr Leu Tyr Ala IleAsn Leu His Asn Val Thr Lys Tyr 450 455 460 ttg cgg tta ccc tat cct ttttct aac aag caa gtg gat aaa tac ctt 1499 Leu Arg Leu Pro Tyr Pro Phe SerAsn Lys Gln Val Asp Lys Tyr Leu 465 470 475 cta aga cct ttg gga cct catgga tta ctt tcc aaa tct gtc caa ctc 1547 Leu Arg Pro Leu Gly Pro His GlyLeu Leu Ser Lys Ser Val Gln Leu 480 485 490 495 aat ggt cta act cta aagatg gtg gat gat caa acc ttg cca cct tta 1595 Asn Gly Leu Thr Leu Lys MetVal Asp Asp Gln Thr Leu Pro Pro Leu 500 505 510 atg gaa aaa cct ctc cggcca gga agt tca ctg ggc ttg cca gct ttc 1643 Met Glu Lys Pro Leu Arg ProGly Ser Ser Leu Gly Leu Pro Ala Phe 515 520 525 tca tat agt ttt ttt gtgata aga aat gcc aaa gtt gct gct tgc atc 1691 Ser Tyr Ser Phe Phe Val IleArg Asn Ala Lys Val Ala Ala Cys Ile 530 535 540 tgaaaataaa atatactagtcctgacactg 1721 12 824 DNA Mus musculus 12 ctggcaagaa ggtctggttgggagagacga gctcagctta cggtggcggt gcacccttgc 60 tgtccaacac ctttgcagctggctttatgt ggctggataa attgggcctg tcagcccaga 120 tgggcataga agtcgtgatgaggcaggtgt tcttcggagc aggcaactac cacttagtgg 180 atgaaaactt tgagcctttacctgattact ggctctctct tctgttcaag aaactggtag 240 gtcccagggt gttactgtcaagagtgaaag gcccagacag gagcaaactc cgagtgtatc 300 tccactgcac taacgtctatcacccacgat atcaggaagg agatctaact ctgtatgtcc 360 tgaacctcca taatgtcaccaagcacttga aggtaccgcc tccgttgttc aggaaaccag 420 tggatacgta ccttctgaagccttcggggc cggatggatt actttccaaa tctgtccaac 480 tgaacggtca aattctgaagatggtggatg agcagaccct gccagctttg acagaaaaac 540 ctctccccgc aggaagtgcactaagcctgc ctgccttttc ctatggtttt tttgtcataa 600 gaaatgccaa aatcgctgcttgtatatgaa aataaaaggc atacggtacc cctgagacaa 660 aagccgaggg gggtgttattcataaaacaa aaccctagtt taggaggcca cctccttgcc 720 gagttccaga gcttcgggagggtggggtac acttcagtat tacattcagt gtggtgttct 780 ctctaagaag aatactgcaggtggtgacag ttaatagcac tgtg 824 13 1899 DNA Homo sapiens 13 gggaaagcgagcaaggaagt aggagagagc cgggcaggcg gggcggggtt ggattgggag 60 cagtgggagggatgcagaag aggagtggga gggatggagg gcgcagtggg aggggtgagg 120 aggcgtaacggggcggagga aaggagaaaa gggcgctggg gctcggcggg aggaagtgct 180 agagctctcgactctccgct gcgcggcagc tggcgggggg agcagccagg tgagcccaag 240 atgctgctgcgctcgaagcc tgcgctgccg ccgccgctga tgctgctgct cctggggccg 300 ctgggtcccctctcccctgg cgccctgccc cgacctgcgc aagcacagga cgtcgtggac 360 ctggacttcttcacccagga gccgctgcac ctggtgagcc cctcgttcct gtccgtcacc 420 attgacgccaacctggccac ggacccgcgg ttcctcatcc tcctgggttc tccaaagctt 480 cgtaccttggccagaggctt gtctcctgcg tacctgaggt ttggtggcac caagacagac 540 ttcctaattttcgatcccaa gaaggaatca acctttgaag agagaagtta ctggcaatct 600 caagtcaaccaggatatttg caaatatgga tccatccctc ctgatgtgga ggagaagtta 660 cggttggaatggccctacca ggagcaattg ctactccgag aacactacca gaaaaagttc 720 aagaacagcacctactcaag aagctctgta gatgtgctat acacttttgc aaactgctca 780 ggactggacttgatctttgg cctaaatgcg ttattaagaa cagcagattt gcagtggaac 840 agttctaatgctcagttgct cctggactac tgctcttcca aggggtataa catttcttgg 900 gaactaggcaatgaacctaa cagtttcctt aagaaggctg atattttcat caatgggtcg 960 cagttaggagaagattatat tcaattgcat aaacttctaa gaaagtccac cttcaaaaat 1020 gcaaaactctatggtcctga tgttggtcag cctcgaagaa agacggctaa gatgctgaag 1080 agcttcctgaaggctggtgg agaagtgatt gattcagtta catggcatca ctactatttg 1140 aatggacggactgctaccag ggaagatttt ctaaaccctg atgtattgga catttttatt 1200 tcatctgtgcaaaaagtttt ccaggtggtt gagagcacca ggcctggcaa gaaggtctgg 1260 ttaggagaaacaagctctgc atatggaggc ggagcgccct tgctatccga cacctttgca 1320 gctggctttatgtggctgga taaattgggc ctgtcagccc gaatgggaat agaagtggtg 1380 atgaggcaagtattctttgg agcaggaaac taccatttag tggatgaaaa cttcgatcct 1440 ttacctgattattggctatc tcttctgttc aagaaattgg tgggcaccaa ggtgttaatg 1500 gcaagcgtgcaaggttcaaa gagaaggaag cttcgagtat accttcattg cacaaacact 1560 gacaatccaaggtataaaga aggagattta actctgtatg ccataaacct ccataacgtc 1620 accaagtacttgcggttacc ctatcctttt tctaacaagc aagtggataa ataccttcta 1680 agacctttgggacctcatgg attactttcc aaatctgtcc aactcaatgg tctaactcta 1740 aagatggtggatgatcaaac cttgccacct ttaatggaaa aacctctccg gccaggaagt 1800 tcactgggcttgccagcttt ctcatatagt ttttttgtga taagaaatgc caaagttgct 1860 gcttgcatctgaaaataaaa tatactagtc ctgacactg 1899 14 592 PRT Homo sapiens 14 Met GluGly Ala Val Gly Gly Val Arg Arg Arg Asn Gly Ala Glu Glu 1 5 10 15 ArgArg Lys Gly Arg Trp Gly Ser Ala Gly Gly Ser Ala Arg Ala Leu 20 25 30 AspSer Pro Leu Arg Gly Ser Trp Arg Gly Glu Gln Pro Gly Glu Pro 35 40 45 LysMet Leu Leu Arg Ser Lys Pro Ala Leu Pro Pro Pro Leu Met Leu 50 55 60 LeuLeu Leu Gly Pro Leu Gly Pro Leu Ser Pro Gly Ala Leu Pro Arg 65 70 75 80Pro Ala Gln Ala Gln Asp Val Val Asp Leu Asp Phe Phe Thr Gln Glu 85 90 95Pro Leu His Leu Val Ser Pro Ser Phe Leu Ser Val Thr Ile Asp Ala 100 105110 Asn Leu Ala Thr Asp Pro Arg Phe Leu Ile Leu Leu Gly Ser Pro Lys 115120 125 Leu Arg Thr Leu Ala Arg Gly Leu Ser Pro Ala Tyr Leu Arg Phe Gly130 135 140 Gly Thr Lys Thr Asp Phe Leu Ile Phe Asp Pro Lys Lys Glu SerThr 145 150 155 160 Phe Glu Glu Arg Ser Tyr Trp Gln Ser Gln Val Asn GlnAsp Ile Cys 165 170 175 Lys Tyr Gly Ser Ile Pro Pro Asp Val Glu Glu LysLeu Arg Leu Glu 180 185 190 Trp Pro Tyr Gln Glu Gln Leu Leu Leu Arg GluHis Tyr Gln Lys Lys 195 200 205 Phe Lys Asn Ser Thr Tyr Ser Arg Ser SerVal Asp Val Leu Tyr Thr 210 215 220 Phe Ala Asn Cys Ser Gly Leu Asp LeuIle Phe Gly Leu Asn Ala Leu 225 230 235 240 Leu Arg Thr Ala Asp Leu GlnTrp Asn Ser Ser Asn Ala Gln Leu Leu 245 250 255 Leu Asp Tyr Cys Ser SerLys Gly Tyr Asn Ile Ser Trp Glu Leu Gly 260 265 270 Asn Glu Pro Asn SerPhe Leu Lys Lys Ala Asp Ile Phe Ile Asn Gly 275 280 285 Ser Gln Leu GlyGlu Asp Tyr Ile Gln Leu His Lys Leu Leu Arg Lys 290 295 300 Ser Thr PheLys Asn Ala Lys Leu Tyr Gly Pro Asp Val Gly Gln Pro 305 310 315 320 ArgArg Lys Thr Ala Lys Met Leu Lys Ser Phe Leu Lys Ala Gly Gly 325 330 335Glu Val Ile Asp Ser Val Thr Trp His His Tyr Tyr Leu Asn Gly Arg 340 345350 Thr Ala Thr Arg Glu Asp Phe Leu Asn Pro Asp Val Leu Asp Ile Phe 355360 365 Ile Ser Ser Val Gln Lys Val Phe Gln Val Val Glu Ser Thr Arg Pro370 375 380 Gly Lys Lys Val Trp Leu Gly Glu Thr Ser Ser Ala Tyr Gly GlyGly 385 390 395 400 Ala Pro Leu Leu Ser Asp Thr Phe Ala Ala Gly Phe MetTrp Leu Asp 405 410 415 Lys Leu Gly Leu Ser Ala Arg Met Gly Ile Glu ValVal Met Arg Gln 420 425 430 Val Phe Phe Gly Ala Gly Asn Tyr His Leu ValAsp Glu Asn Phe Asp 435 440 445 Pro Leu Pro Asp Tyr Trp Leu Ser Leu LeuPhe Lys Lys Leu Val Gly 450 455 460 Thr Lys Val Leu Met Ala Ser Val GlnGly Ser Lys Arg Arg Lys Leu 465 470 475 480 Arg Val Tyr Leu His Cys ThrAsn Thr Asp Asn Pro Arg Tyr Lys Glu 485 490 495 Gly Asp Leu Thr Leu TyrAla Ile Asn Leu His Asn Val Thr Lys Tyr 500 505 510 Leu Arg Leu Pro TyrPro Phe Ser Asn Lys Gln Val Asp Lys Tyr Leu 515 520 525 Leu Arg Pro LeuGly Pro His Gly Leu Leu Ser Lys Ser Val Gln Leu 530 535 540 Asn Gly LeuThr Leu Lys Met Val Asp Asp Gln Thr Leu Pro Pro Leu 545 550 555 560 MetGlu Lys Pro Leu Arg Pro Gly Ser Ser Leu Gly Leu Pro Ala Phe 565 570 575Ser Tyr Ser Phe Phe Val Ile Arg Asn Ala Lys Val Ala Ala Cys Ile 580 585590 15 1899 DNA Homo sapiens CDS (94)..(1869) 15 gggaaagcga gcaaggaagtaggagagagc cgggcaggcg gggcggggtt ggattgggag 60 cagtgggagg gatgcagaagaggagtggga ggg atg gag ggc gca gtg gga ggg 114 Met Glu Gly Ala Val GlyGly 1 5 gtg agg agg cgt aac ggg gcg gag gaa agg aga aaa ggg cgc tgg ggc162 Val Arg Arg Arg Asn Gly Ala Glu Glu Arg Arg Lys Gly Arg Trp Gly 1015 20 tcg gcg gga gga agt gct aga gct ctc gac tct ccg ctg cgc ggc agc210 Ser Ala Gly Gly Ser Ala Arg Ala Leu Asp Ser Pro Leu Arg Gly Ser 2530 35 tgg cgg ggg gag cag cca ggt gag ccc aag atg ctg ctg cgc tcg aag258 Trp Arg Gly Glu Gln Pro Gly Glu Pro Lys Met Leu Leu Arg Ser Lys 4045 50 55 cct gcg ctg ccg ccg ccg ctg atg ctg ctg ctc ctg ggg ccg ctg ggt306 Pro Ala Leu Pro Pro Pro Leu Met Leu Leu Leu Leu Gly Pro Leu Gly 6065 70 ccc ctc tcc cct ggc gcc ctg ccc cga cct gcg caa gca cag gac gtc354 Pro Leu Ser Pro Gly Ala Leu Pro Arg Pro Ala Gln Ala Gln Asp Val 7580 85 gtg gac ctg gac ttc ttc acc cag gag ccg ctg cac ctg gtg agc ccc402 Val Asp Leu Asp Phe Phe Thr Gln Glu Pro Leu His Leu Val Ser Pro 9095 100 tcg ttc ctg tcc gtc acc att gac gcc aac ctg gcc acg gac ccg cgg450 Ser Phe Leu Ser Val Thr Ile Asp Ala Asn Leu Ala Thr Asp Pro Arg 105110 115 ttc ctc atc ctc ctg ggt tct cca aag ctt cgt acc ttg gcc aga ggc498 Phe Leu Ile Leu Leu Gly Ser Pro Lys Leu Arg Thr Leu Ala Arg Gly 120125 130 135 ttg tct cct gcg tac ctg agg ttt ggt ggc acc aag aca gac ttccta 546 Leu Ser Pro Ala Tyr Leu Arg Phe Gly Gly Thr Lys Thr Asp Phe Leu140 145 150 att ttc gat ccc aag aag gaa tca acc ttt gaa gag aga agt tactgg 594 Ile Phe Asp Pro Lys Lys Glu Ser Thr Phe Glu Glu Arg Ser Tyr Trp155 160 165 caa tct caa gtc aac cag gat att tgc aaa tat gga tcc atc cctcct 642 Gln Ser Gln Val Asn Gln Asp Ile Cys Lys Tyr Gly Ser Ile Pro Pro170 175 180 gat gtg gag gag aag tta cgg ttg gaa tgg ccc tac cag gag caattg 690 Asp Val Glu Glu Lys Leu Arg Leu Glu Trp Pro Tyr Gln Glu Gln Leu185 190 195 cta ctc cga gaa cac tac cag aaa aag ttc aag aac agc acc tactca 738 Leu Leu Arg Glu His Tyr Gln Lys Lys Phe Lys Asn Ser Thr Tyr Ser200 205 210 215 aga agc tct gta gat gtg cta tac act ttt gca aac tgc tcagga ctg 786 Arg Ser Ser Val Asp Val Leu Tyr Thr Phe Ala Asn Cys Ser GlyLeu 220 225 230 gac ttg atc ttt ggc cta aat gcg tta tta aga aca gca gatttg cag 834 Asp Leu Ile Phe Gly Leu Asn Ala Leu Leu Arg Thr Ala Asp LeuGln 235 240 245 tgg aac agt tct aat gct cag ttg ctc ctg gac tac tgc tcttcc aag 882 Trp Asn Ser Ser Asn Ala Gln Leu Leu Leu Asp Tyr Cys Ser SerLys 250 255 260 ggg tat aac att tct tgg gaa cta ggc aat gaa cct aac agtttc ctt 930 Gly Tyr Asn Ile Ser Trp Glu Leu Gly Asn Glu Pro Asn Ser PheLeu 265 270 275 aag aag gct gat att ttc atc aat ggg tcg cag tta gga gaagat tat 978 Lys Lys Ala Asp Ile Phe Ile Asn Gly Ser Gln Leu Gly Glu AspTyr 280 285 290 295 att caa ttg cat aaa ctt cta aga aag tcc acc ttc aaaaat gca aaa 1026 Ile Gln Leu His Lys Leu Leu Arg Lys Ser Thr Phe Lys AsnAla Lys 300 305 310 ctc tat ggt cct gat gtt ggt cag cct cga aga aag acggct aag atg 1074 Leu Tyr Gly Pro Asp Val Gly Gln Pro Arg Arg Lys Thr AlaLys Met 315 320 325 ctg aag agc ttc ctg aag gct ggt gga gaa gtg att gattca gtt aca 1122 Leu Lys Ser Phe Leu Lys Ala Gly Gly Glu Val Ile Asp SerVal Thr 330 335 340 tgg cat cac tac tat ttg aat gga cgg act gct acc agggaa gat ttt 1170 Trp His His Tyr Tyr Leu Asn Gly Arg Thr Ala Thr Arg GluAsp Phe 345 350 355 cta aac cct gat gta ttg gac att ttt att tca tct gtgcaa aaa gtt 1218 Leu Asn Pro Asp Val Leu Asp Ile Phe Ile Ser Ser Val GlnLys Val 360 365 370 375 ttc cag gtg gtt gag agc acc agg cct ggc aag aaggtc tgg tta gga 1266 Phe Gln Val Val Glu Ser Thr Arg Pro Gly Lys Lys ValTrp Leu Gly 380 385 390 gaa aca agc tct gca tat gga ggc gga gcg ccc ttgcta tcc gac acc 1314 Glu Thr Ser Ser Ala Tyr Gly Gly Gly Ala Pro Leu LeuSer Asp Thr 395 400 405 ttt gca gct ggc ttt atg tgg ctg gat aaa ttg ggcctg tca gcc cga 1362 Phe Ala Ala Gly Phe Met Trp Leu Asp Lys Leu Gly LeuSer Ala Arg 410 415 420 atg gga ata gaa gtg gtg atg agg caa gta ttc tttgga gca gga aac 1410 Met Gly Ile Glu Val Val Met Arg Gln Val Phe Phe GlyAla Gly Asn 425 430 435 tac cat tta gtg gat gaa aac ttc gat cct tta cctgat tat tgg cta 1458 Tyr His Leu Val Asp Glu Asn Phe Asp Pro Leu Pro AspTyr Trp Leu 440 445 450 455 tct ctt ctg ttc aag aaa ttg gtg ggc acc aaggtg tta atg gca agc 1506 Ser Leu Leu Phe Lys Lys Leu Val Gly Thr Lys ValLeu Met Ala Ser 460 465 470 gtg caa ggt tca aag aga agg aag ctt cga gtatac ctt cat tgc aca 1554 Val Gln Gly Ser Lys Arg Arg Lys Leu Arg Val TyrLeu His Cys Thr 475 480 485 aac act gac aat cca agg tat aaa gaa gga gattta act ctg tat gcc 1602 Asn Thr Asp Asn Pro Arg Tyr Lys Glu Gly Asp LeuThr Leu Tyr Ala 490 495 500 ata aac ctc cat aac gtc acc aag tac ttg cggtta ccc tat cct ttt 1650 Ile Asn Leu His Asn Val Thr Lys Tyr Leu Arg LeuPro Tyr Pro Phe 505 510 515 tct aac aag caa gtg gat aaa tac ctt cta agacct ttg gga cct cat 1698 Ser Asn Lys Gln Val Asp Lys Tyr Leu Leu Arg ProLeu Gly Pro His 520 525 530 535 gga tta ctt tcc aaa tct gtc caa ctc aatggt cta act cta aag atg 1746 Gly Leu Leu Ser Lys Ser Val Gln Leu Asn GlyLeu Thr Leu Lys Met 540 545 550 gtg gat gat caa acc ttg cca cct tta atggaa aaa cct ctc cgg cca 1794 Val Asp Asp Gln Thr Leu Pro Pro Leu Met GluLys Pro Leu Arg Pro 555 560 565 gga agt tca ctg ggc ttg cca gct ttc tcatat agt ttt ttt gtg ata 1842 Gly Ser Ser Leu Gly Leu Pro Ala Phe Ser TyrSer Phe Phe Val Ile 570 575 580 aga aat gcc aaa gtt gct gct tgc atctgaaaataaa atatactagt 1889 Arg Asn Ala Lys Val Ala Ala Cys Ile 585 590cctgacactg 1899 16 594 DNA Homo sapiens 16 attactatag ggcacgcgtggtcgacggcc cgggctggta ttgtcttaat gagaagttga 60 taaagaattt tgggtggttgatctctttcc agctgcagtt tagcgtatgc tgaggccaga 120 ttttttcagg caaaagtaaaatacctgaga aactgcctgg ccagaggaca atcagatttt 180 ggctggctca agtgacaagcaagtgtttat aagctagatg ggagaggaag ggatgaatac 240 tccattggag gctttactcgagggtcagag ggatacccgg cgccatcaga atgggatctg 300 ggagtcggaa acgctgggttcccacgagag cgcgcagaac acgtgcgtca ggaagcctgg 360 tccgggatgc ccagcgctgctccccgggcg ctcctccccg ggcgctcctc cccaggcctc 420 ccgggcgctt ggatcccggccatctccgca cccttcaagt gggtgtgggt gatttcgtaa 480 gtgaacgtga ccgccaccggggggaaagcg agcaaggaag taggagagag ccgggcaggc 540 ggggcggggt tggattgggagcagtgggag ggatgcagaa gaggagtggg aggg 594 17 21 DNA Artificial sequenceSingle strand DNA oligonucleotide 17 ccccaggagc agcagcatca g 21 18 21DNA Artificial sequence Single strand DNA oligonucleotide 18 aggcttcgagcgcagcagca t 21 19 22 DNA Artificial sequence Single strand DNAoligonucleotide 19 gtaatacgac tcactatagg gc 22 20 19 DNA Artificialsequence Single strand DNA oligonucleotide 20 actatagggc acgcgtggt 19 2121 DNA Artificial sequence Single strand DNA oligonucleotide 21cttgggctca cctggctgct c 21 22 23 DNA Artificial sequence Single strandDNA oligonucleotide 22 agctctgtag atgtgctata cac 23 23 22 DNA Artificialsequence Single strand DNA oligonucleotide 23 gcatcttagc cgtctttctt cg22 24 23 DNA Artificial sequence Single strand DNA oligonucleotide 24gagcagccag gtgagcccaa gat 23 25 23 DNA Artificial sequence Single strandDNA oligonucleotide 25 ttcgatccca agaaggaatc aac 23 26 23 DNA Artificialsequence Single strand DNA oligonucleotide 26 agctctgtag atgtgctata cac23 27 24 DNA Artificial sequence Single strand DNA oligonucleotide 27tcagatgcaa gcagcaactt tggc 24 28 22 DNA Artificial sequence Singlestrand DNA oligonucleotide 28 gcatcttagc cgtctttctt cg 22 29 24 DNAArtificial sequence Single strand DNA oligonucleotide 29 gtagtgatgccatgtaactg aatc 24 30 22 DNA Artificial sequence Single strand DNAoligonucleotide 30 aggcacccta gagatgttcc ag 22 31 24 DNA Artificialsequence Single strand DNA oligonucleotide 31 gaagatttct gtttccatga cgtg24 32 25 DNA Artificial sequence Single strand DNA oligonucleotide 32ccacactgaa tgtaatactg aagtg 25 33 22 DNA Artificial sequence Singlestrand DNA oligonucleotide 33 cgaagctctg gaactcggca ag 22 34 22 DNAArtificial sequence Single strand DNA oligonucleotide 34 gccagctgcaaaggtgttgg ac 22 35 23 DNA Artificial sequence Single strand DNAoligonucleotide 35 aacacctgcc tcatcacgac ttc 23 36 22 DNA Artificialsequence Single strand DNA oligonucleotide 36 gccaggctgg cgtcgatggt ga22 37 22 DNA Artificial sequence Single strand DNA oligonucleotide 37gtcgatggtg atggacagga ac 22 38 22 DNA Artificial sequence Single strandDNA oligonucleotide 38 gtaatacgac tcactatagg gc 22 39 19 DNA Artificialsequence Single strand DNA oligonucleotide 39 actatagggc acgcgtggt 19 4027 DNA Artificial sequence Single strand DNA oligonucleotide 40ccatcctaat acgactcact atagggc 27 41 23 DNA Artificial sequence Singlestrand DNA oligonucleotide 41 actcactata gggctcgagc ggc 23 42 44848 DNAHomo sapiens 42 ggatcttggc tcactgcaat ctctgcctcc catgcaattc ttatgcatcagcctcctgag 60 tagcttggat tataggtctg cgccaccact cctggctaca ccatgttgcccaggctggtc 120 ttgaactctt gggctctagt gatccacccg ccttggcctc ccaaagtgctgggattacag 180 gtgtgagcca tcacacccgg ccccccgttt ccatattagt aactcacatgtagaccacaa 240 ggatgcacta tttagaaaac ttgcaatggt ccacttttca aatcacccaaacatgttaaa 300 gaaattggta tgactgggca tggcacagtg gctcatgcct gcaatcctagcattttgtga 360 ggctgagacg ggcagatcac gaggtcagga gattgagacc atcctgacagacatggtgaa 420 atcccatctc tactaaaaat acaaaacaat tagccggggg tgatggcaggcccctgtagt 480 cccagctact cgggaggctg aggcaggaga atggcgtgaa tccaggaggcagagcttgca 540 gtgagccgag atggtgccac tgcactccag cctgggcgac agagcgagactccgtctcaa 600 aaaaaaaaaa aaagaaagaa attggtatga ctgttgactc acaacaggagtcaggggcat 660 ggggtggggt gtaagattaa tgtcatgaca aatgtggaaa agaaacttctgtttttccaa 720 ctccacgtct gctaccatat tattacactc ttctggtagt gtggtgtttatgtgtgaatt 780 ttttttcata tgtatacagt aattgtagga tatgaacctg attctagttgcaaaactcac 840 tatgagctta gcttttaagt tgcttaagaa taggtagatc tatgcaaataatgataatta 900 ttattattat tttaagagag ggtctcactt tgtcacccag gctggagtgcagtggtgtga 960 ttaagggtca ctgcaacctc cacctcccag gctcaaataa acctcccacctcagcctccc 1020 cagtagctgg aaccacaggc acgggccacc acgcctggct aattttttgtattttttgta 1080 gagatggggt ttcatcatgt tgcccaggct gttcttgaat tcctcggctcaagcaatcct 1140 cccaccttgg cctcccaaaa tgctggcatc acaggcatga tggcatcactggcatcacat 1200 accatgcctg gcctgattta tgcaaattag atatgcattt caaaataatctatttttatt 1260 tgttgcctta ttggtggtac aatctcaagt ggaaaaatct aagggttttggtgttatttg 1320 cttactcaac caatatttat tagactctta ctaagcacca acatgatcacatgcctgagc 1380 tatggctagc atagcgtgtg agacaaactt aatctctgtt ttggtggagcatataatcta 1440 gtagatgaag ccaatgttga gcaacatcac aatactaaca aattgaggatgctacgagag 1500 tgtctaacaa attgaggatg ctacgagagt gtctaacaaa ttgaggatgctatgagagtg 1560 tgtcatggag agctgcctgg agattgagag aaagcttcct tgagggaagttacatttcag 1620 ctgaaacaca ctgccatctg ctcgaggttt tgtaactgca ttcacatcccgattctgaca 1680 cttcacatcc cgattctgac acttcaccca gttactgtct cagagcttgggtccgcatgt 1740 gtaaaacaag gacagtatgc acttggcagg gttgtgagaa gggaagagaacacaagtaaa 1800 gcacctgtat caggcataca gtaggcacta agcgtgcgat gcttgctatgattatacatc 1860 agtgtaagca tcaaggaaaa gctgaagaaa agtctgacca acagcgaaagataaatgcgc 1920 agaggagaaa tttggcaaag gctccaaatt caggggcagt ccgtactctacactttgtat 1980 gggggcttca ggtcctgagt tccagacatt ggagcaacta accctttaagattgctaaat 2040 attgtcttaa tgagaagttg ataaagaatt ttgggtggtt gatctctttccagctgcagt 2100 ttagcgtatg ctgaggccag attttttcaa gcaaaagtaa aatacctgagaaactgcctg 2160 gccagaggac aatcagattt tggctggctc aagtgacaag caagtgtttataagctagat 2220 gggagaggaa gggatgaata ctccattgga ggttttactc gagggtcagagggatacccg 2280 gcgccatcag aatgggatct gggagtcgga aacgctgggt tcccacgagagcgcgcagaa 2340 cacgtgcgtc aggaagcctg gtccgggatg cccagcgctg ctccccgggcgctcctcccc 2400 gggcgctcct ccccaggcct cccgggcgct tggatcccgg ccatctccgcacccttcaag 2460 tgggtgtggg tgatttcgta agtgaacgtg accgccaccg aggggaaagcgagcaaggaa 2520 gtaggagaga gccgggcagg cggggcgggg ttggattggg agcagtgggagggatgcaga 2580 agaggagtgg gagggatgga gggcgcagtg ggaggggtga ggaggcgtaacggggcggag 2640 gaaaggagaa aagggcgctg gggctcggcg ggaggaagtg ctagagctctcgactctccg 2700 ctgcgcggca gctggcgggg ggagcagcca ggtgagccca agatgctgctgcgctcgaag 2760 cctgcgctgc cgccgccgct gatgctgctg ctcctggggc cgctgggtcccctctcccct 2820 ggcgccctgc cccgacctgc gcaagcacag gacgtcgtgg acctggacttcttcacccag 2880 gagccgctgc acctggtgag cccctcgttc ctgtccgtca ccattgacgccaacctggcc 2940 acggacccgc ggttcctcat cctcctgggg taagcgccag cctcctggtcctgtcccctt 3000 tcctgtcctc ctgacaccta tgtctgcccc gccagcggct ctccttcttttgcgcggaaa 3060 caacttcaca ccggaacctc cccgcctgtc tctccccacc ccacttcccgcctctcattc 3120 tccctctccc tcccttactc tcagacccca aaccgctttt tggggggtatcatttaaaaa 3180 atagatttag gggttacaag tgcagttctg ttccatgggt atattgcattgtggtggcat 3240 ctgggctctt agtgtaactg tcacccgaat gttgtacatt gtatctaataggtaatttct 3300 catccctcat ccctctccca ccctcccacc ttttggagtc tccagtgtctactattccac 3360 taagtccatg tgtacacatt gtttagcgcc cactctaaat gagcctttttgtttcattca 3420 ttctgtaagt gttgaatagg caccacctaa ggtcaggtat aagtggaaatttgaaaaaga 3480 aactgcccac ttgccccagt acttccctag ccaagaggag ggaaaccaggcaggtgcacc 3540 tgaaggcctg tgagtgcttg atttgctgtg cagtgtagga caagtaagattgtgcatagc 3600 cttctgtatt taagactgtg ttaggaagat ttctctttct tttcttttctttttcttttt 3660 tcttttcttt ttttttttta ggcagatgaa aagggcgtca cagaacaggaataaaaatct 3720 aaatattcaa taaatgagac ctaggagact actgcagtga cttacaaagtcctaataaaa 3780 agatgtctct ccaaaatggg gctgcaaaat gtggtgctgc cttatcagctctaagttttt 3840 tccttacctg agaaagaagg aacctgatgc aggttcaggg ctcctgccccatgaatgcag 3900 gctgactcca agatggggag ctacagggac aatcccaggt cttctaggcctcttatttag 3960 gccctgggag cctccagaga tggccacatc ttgaccagcc cagatagagggaaagatcac 4020 cattatctca cctctgtgtc aaatacctag atgctgtcct ccctgagcccacactatagt 4080 tgccagcgct aatttaatgg gtagtgtact ggttaagaga tggacagaccatcctggctt 4140 gactctcagc tctggcaaag atgagtgact tggtttttcc atatctcttggccacaccaa 4200 ccttgatttc ttcagctgta gaatggaatt tctcaagctt gcctcaaggattattgcccg 4260 aggatttgat gatatggtaa gagcttctca gtgtttgacc catagtaagtgtttgacgtt 4320 tcaaacgaat tgtttctttc taggacatgg tgagcatttg gtagccattcaccggttttc 4380 tgtttctttg gatcatagtt aacctctcct tttccttctg gcactacaattttctggtgg 4440 ggaagaatcc ttactttctg cccttcccct taaggatagg aagctgatactaggcagcaa 4500 ctagttgggg gataggaaga ttgttccaga gaaatgctga accatagggctccagatcac 4560 aggaccccag tcttagcttg ctggggtgtg gggtgggggg gggcggttactgaacatggg 4620 tatgaagtag atgtccattt actgaaatgt gaggacctga ggcctcttctattgctgtag 4680 ccagcatatt ccccaacctc tccccaagaa aggacagatg ggggttcccccctggagtaa 4740 caggtccaaa agaaaaaaca tacagtggga cttccaggat ctgggcctgatcacccagca 4800 gtcaagctcc ccgcaattga ctaacacccc cctaacacgt agaaattccaatctgcaatt 4860 tagtgaggat gataccttta ttcttcttaa atacatctct tcatttcccagagcaccctt 4920 ttttcccctc ctctgcacct ttttgttaaa gactggagta taatgaaataccaagagagc 4980 ataacatgtg atacataaaa ctttttttct ggtttacaaa acagttcattcttgtccata 5040 cgtgcttctc tccaaggctg gctgctgtct gttccagccc gcttcgcttggagaggccat 5100 ctgccatacc tgctccccag acgcatcgac aagcacaccc agagtgttatctgctaagac 5160 ctaaaagagg gaggaacccc ctctcctcat ctaagaccta gcttctaaattagagtgtga 5220 gggtccatct ccccaggagg ggcacagggc ccaaacagcc cagccatctcagaagacaac 5280 actaagcttt gtaggggtcc acagtagagg agagtaagac gcctgttgtttaatttatta 5340 cagttcctca aaagtgaaga tgtgtgggcg ggatggcaag agctgagcagacgaaagctg 5400 aaggaataag gaaagagagg aggacacaaa cagctgacac ttcctcagttcttgtcattt 5460 gcctggccct gttctaagca ccttctaggt attaatccat ttagtcttggctacaacact 5520 gtgagtaact agttttgtca cccccatttt aaaaatgaag aaagtgaggctcagggaggt 5580 taagtaactt ggccacagtt tgaaactaga ctctgatcac atgagataatagtgcccata 5640 aaaagggaaa gcagattata ttttttaaag gaaagagagt aggatatggtagaaaaagat 5700 tgtttggaaa ggaattgaga gattgatata atgaaaagaa gcattcacatgagagtaaca 5760 gtatcagggc ccaaaccttc atctaaggta cttcaaagag gcctaagcaaacttagtcac 5820 tggcgtggtt ctagtctcca tgatggcaaa tacattgtgt acagcccaactccacacaaa 5880 acttaaatac caatgataga gcaatctaaa atttgaaaga aaaaatctttcaatttgtcg 5940 tcttcccaga gggacttaat caagaaacca atcaaaatac ttcctaagcctaactgtgtg 6000 cagaactcca aagagagccc agccctaaat caacactgtc caatggaaatataatataat 6060 gtgggcctca tatgcaaggt catatgtaat tttaaatttt ctagtagccatattaaaaag 6120 gtaaaaagaa acaagtgaaa ttaattttaa taattttatt tagttcaatagatccaaaat 6180 gttttctcag catgtaatca atataaaaat attaatgagg tatttattattccttttctc 6240 aaaccaagtc tattctataa tctggcgtgt attatttaca gcacttctcagactatattt 6300 ctttctttct tttttttttc cgagacaatt ttgctcttgt cacccaagctagagtacaat 6360 ggcgttacct cggctcactg caacctccgc ctcccgggtt caagttattctcctgcctca 6420 gtctcccaag tagctgggac tagaggcatg caccaccacg cctggctaattgtgtatttt 6480 tagtagagac agggtttcac catgttggcc aggctaatct caaactcctgagctcaggtg 6540 atatgcccac ctcggcctcc caaagtgttg ggattacagg cgtgagccactgcacccggc 6600 ctcagattaa ctatatttca agcgttcagt agccacatgt agctagtgctatggtagtgg 6660 acagtacaga tctgcatttc aattaagaca cgtatacaag catagttcactaatgcacgg 6720 taaaaaaaag tatagtgctg agtcggtggt agaaatccta aatactgcagagcaaaagtg 6780 gtacgaacag caatctcagt gataatgcaa ccatgcttgc ttttcattgcaatttgctta 6840 ttttccttca gcaaagttca tccatttttg ccaattcaat aaatatttactgataaaaac 6900 tttcaatatt agattcttgc atcttcatag acagagttgc ttttcacatttagaaaatta 6960 cttatcaatg ttaaacacac gttttgataa ccagtgttgg aaagaggtgcagactcccca 7020 tgtgcctatt gatggcagaa atattcacag ccaaagggaa acaaagggctggggacaatc 7080 acacacctca tgtctcctaa ctcctgggaa gtgctgtccc tctgattgagctcttattat 7140 tgccttcccc actaaccctg tccactgtgc cctggagccc tttgcagggttacctgctct 7200 gtcctcctca cagaatatct cctctacctc cttgtccaag ctacaacttggctattctct 7260 gatgacactg tcttccctgt agcccttttg agtaatggct gcatattctcccatagtcca 7320 gttcttttcc tgttctccag tctggcttct ggatgacagc ccactagtttgaactccata 7380 ctgctatagt tcaagtccct tttgacttgt taccttgggc aaattacctccttttgttca 7440 ggttccttgt ttgtaaaatg acgataataa tgccatttgc ttcagtgggttattttgaaa 7500 ttgagtgaaa gaaggcgggt agcttcccta cacgctcagt gtagactagcctgatgtgca 7560 ttacgggtga tgccatgact cagtgtgttt tcctcatctc cacatctggctctcatccag 7620 tgctcctgct tacggcactc tgtccccctc ttacttactc ccccttattaactgaagact 7680 ggcactgatc tcacagtttc ctctccactt cctagtctca ccatcatcctagatgacttc 7740 aagtcaccta gataaactgt ctcagtttct tcactcacat ttttttataacagataatgt 7800 tacactcaag ttgtaacaga accagcttat ccagctcatg aaatgtatgcatttcatctc 7860 aactctgtat tcagtgacat cctgtgggta tctggaaatc agccatggtgagaatattta 7920 ccatggaaat tggcaaatac taaaaagcag agcacctttt tttctgagagccagaccata 7980 gctcttctac tccatagcac ccatcataac aatttttaaa tacctccactgaacagcttc 8040 ttcctctctc tacttcttcc atatctgatt tgagcttctt aatttatcatgtgaaccact 8100 cttgtaataa taaccccaaa tccctgttcc attgttcttc ctgctaaaatactaaacctg 8160 gtttagtcca accatatttt ctctctttgg aatctacagg gtggcccaaaaacctggaaa 8220 tggaaaaata ttacttatta attttaatgt atattaataa gccattttaatgcttcattt 8280 ccagtctcag tggccaccct gtatagctgg gctattgagc tcttgcgggaggagggagtg 8340 gacagtctcc cagccacaca gactgatgtt gcaccaaaca ttttttagcttccagacttc 8400 cctggccctt agtgttaccc ttaactctcc atttctctgc ctttcacattctctactttt 8460 taaaaatctc tgactccacc ttcaccttat cattcttagc acatgaccatacttctgctt 8520 cccaaagaaa atgagcaatt acttcctttt ccttttcctc ctgtcatcaaatctgcagac 8580 atgtcatgcc taagtccagc tttcctcctt tctctgatct cagtctgcttcttccatttc 8640 tgccctgaat cccgtcccct ccccaacccc caaggacttc gctctatcagtcacctcttc 8700 cctctcctgt atcttcaact cctcccattt tactggcttc ttcctcaagcctttccccaa 8760 gcctttccca tctcaattac ctcctcgcac atgcctctgc agaaaccaccccgtttcttc 8820 cctcccctcg gcagcctgtt cttcctgttc tgccctcatg atggcaccatcattgtgtca 8880 ctaaaatcaa tctctccgac atcatcaatg gccttccttt gttgggaaacctaataaaca 8940 ctttatctta tttggtcttt gttatgggtt gaatgaggtt accccgaaatccatattaga 9000 agtcctaacc cccagtacct cagaatgtga ctttatttgg gaatagggtcattgcagacg 9060 ttattagtta ggatgaggtc atactggaat gtgatgggct gcttatctaatatgactgat 9120 gtccttataa caaggagaaa tttggagaca gacacgcaca tagggagaataccatgtgat 9180 gacaggagtt atggagttgg agtcaaaaag ctatgggaac ttaggagaaagacctggaac 9240 aaatcctttc ctgcgcctag agagggagta tggccctgcc actaccttgaattcaacgtt 9300 tcggcttttc aaaactgtaa gacaatacat ttctgttgtt caaaccaattagtttgcagt 9360 actctgcgac tgcagcccta acaaactaat acagtctctt ggaggcatttggcaaggttg 9420 acaatggaag cactttctta cccctttagg tctgtcgcct ttcttgttggggggtgtttt 9480 ctaacaattc ctctccatct ctctctctct agtttgtctt aaacattggtgttcttcaga 9540 cttctgacct aggccttctt ttcacttcac atattcccct gggtggtctcacccacttcc 9600 agaaattact taaattactg ctcatgcagt actgtgctgg aaactgtttaacaactggct 9660 ctctgggaag aggggagact ggttgatggt ttttgctgat ttctgtggtgtaaatactcc 9720 ctccatggcc aattccaaac tgccaacagt ttaacaactg gctcacaaattttctccaaa 9780 tttaacattt ggctttcaca ggccaacaac gtggtacagc caactccagcacacctctgc 9840 ttttgtgtca gagagaagta acttattttt gtacaaaagg taaaataaaaacacctgcag 9900 gccccctttt tttccttaac aaactgctct agaaatagaa tagctgaagcttcttttatg 9960 cattcatctg ttatttccat gtcactgtgg tggtgggatt atttttcctttatttttctt 10020 gtatatggtt gaaatactgt acctttgatc agttttagtt ttatggcatgttttgcaccc 10080 atattaaatc tagtttttgt cagagggcgt caatattatt ttctcaaaacaagaaaatat 10140 ttcattgcaa aggagacaaa caaaaaggtc cttaatacca aaactttgaaatgtgatttc 10200 ttgtacttgg cagtgtccaa gtggtaaacc caaacagtat tgggttttcattttgttcag 10260 gaaagtcttt gtctggcagc gacttaccct tacatcaggc gggccttgctcattcattca 10320 cttaagtatt tattaaacac cagcggtgtg ccaagtactt atctaggtatcgggtagatt 10380 ctgataagtc agtcaggtcc ctgctctcag ggagcttgca gcagagatgggggctgcaat 10440 agagagtaag ccaaggaaat gaaaaaggaa gttgatttca gagagtgatgaatgctatga 10500 agaaaatgaa ggcagcgcag tgtgatggag agtgacccaa ggtggtacagtttgtacctc 10560 taaggaccag actgtgaccc aggtcactca cagatgcccg tcatgtgatgccacagcaac 10620 ttttccaggt gctcgtttcc tcccacttcc cagtctcttg cccagccgcgactgcttaca 10680 aatacagcta gaggaatcta aatgaggttc ctctatcatc aaacccaatcaaaatgccaa 10740 ggaacagaat cagtgcctgg ctgaaggcag tggaacaggg ccagcctggagtggttctct 10800 ctgaggaagt tcctcatctt ggttttaggg ccataccttg tgacctgtgagctaggggtt 10860 gccagtccct gacatttcta ctgaggactc gcctgtctat attcccggcctgtatgtgtc 10920 tcctgagttc cagacacaca gggcgaagcg cctgatggat ggaagtatgttttttggtgt 10980 tccattggta tctcaaattc tacaaaactt agtgcccctt ctcctccctgttcctcccca 11040 tcttcagtct atcacctgtt cctcatccag caaatgatat taccatcttccaaggagctt 11100 cccaggagta atccttgact cctcctcaac atccaattaa taatcaaatctaggccaggt 11160 acaatagctc acgcctataa tcccagcact ttgggaggct gaggcaggtggatcatttga 11220 ggccaggagt tcaagaccag cctggccaac aaggtgaaac ctgtctcatttaaaaaaagt 11280 tattttaaaa actcaaatct attatttcta cctctaagtg tgtcttgaatttatccatct 11340 ctctccatct ctgagctgtt accttacctc agtccatcac gttttgtctacgttaacatg 11400 accagagtct tgttcttagt ctggtgaggt cactccagct gcttcagatccttccatggc 11460 tcaccgttgc cctcatataa agttggcact cctggacatg tggcttacggggccctccgt 11520 gatgtggccc tatttgcttc tccattctgt tctctcccag cctctctgcccccatctcta 11580 ggcaccaacc acacccttct gctcgtcaat ggtgccagct tctcttctatctctggtctt 11640 tggacagact tttcccttca cctggaatgc tttcttcaat cctaccccactctctttaat 11700 ctagataagg tttattcttt ttgaatgtct agcagtgaaa ccatttcccctgaaaaacct 11760 tctctaacca accccctacc ctcagcccaa ggtctagatt aggagtccctctgaatgttt 11820 ccatagcatt tttaaagaat tgcctattta cttgttcgta tctatcactaaactacaaat 11880 tgtatgagaa cagccactat ctctgcctgg ttcaccattc atctccagcaactagcataa 11940 tgcctggcag agtcagcctg caacaaatat ttgttgaata aattaacagatggctttatc 12000 tccttaagta aatcttgctt ttttcaccta ttaaaacaga cgcacaggccaggtgtggtg 12060 gcccatgcct gtaatcccag cactttggca ggctgaggtg ggcggatcacctgaggtcag 12120 gagttcaaga ccagcctggc caacatggtg aaaccccatc tctaataaaaatacaaaaat 12180 tagctgggca tggtggtggg tgcgtatagt cccagctact agggaggctgaggcaagaga 12240 atcgcttgaa cccaggaggc agaggtggca gtgagccgag atcatgccactgtactccag 12300 cctggatgac agagaccctg tctcaaaaca cacacacaca cacacacacacacacacaca 12360 cacacacaca cacacacacc aagttgtata atttaaaata taacgtgcttgttatggaac 12420 acttgtaaaa tacaggaaag taatgaaaaa gtctaccatc tagctcaccacataatgacc 12480 attgctatca tcctggcata attctctcct gtatataaat atatattcttttattgttaa 12540 aattacacta tgagtactat ttatttattt tactgtggca aaatgcgcaaaacataaaat 12600 cttgccattt taaggtatgc agtttggtgc attcaccaca ctcacattgttgtgcaaata 12660 tcaccactat ctatctcaga acttcttcgt cttcccaaac tgaaactctgtacccattaa 12720 acaatagtgc atcctctgtt ttcccctccc tacaatttat ttttatttgggtttgtacca 12780 aactgaaaat agctgcttct tccttactta gttcagatta gcatttccatttatttagcc 12840 gtggttttga ggatgccatg acagatgcca tccttcctag agctctttggggctgtcagg 12900 tatttcagtc agggtgaatt cgggttgata acattttaaa atctcactttattctgaggt 12960 tcctagtgtc agagcccacc gtatttttag ggactcccaa gttacaaacaaaaatatggt 13020 gaggaggaat cactgaagtt ttaacacaag agacttacat tttgttcaatttctatcttt 13080 tagtttattt cctaagcata aagaaatact ttgaaaattt tacatagcattatacatatt 13140 taattaagca tgagcacatc ttaaaacttt aaattttaga tcagatctttaattcctagg 13200 atattaagag gtactggcaa tttggccagg tgtggtggtt cacgcctataatcccaacac 13260 tttgggaggg tgaagtgggc gaattgctag agcccaggag gtggaggctgcaatggcctg 13320 agatcacgcc atcgtactcc agcctggatg atgagaatga aatcctgtctcaaaaaaaaa 13380 aaaaaaaaaa aaaagaagaa gaagaagtat tggcaatcag tgctccaggaataatttcct 13440 gacttgaaat aaacctacat gtagacaaac taattaggcc attccaagagttgctagcat 13500 tggtttaata tgttttcaga gcattccagg aagcagtgtg gccagcattgcatgtttgat 13560 acttcagaaa tgtatgacag gtgtttctct tacccaggtc ttctgttttcttagttttgc 13620 tcatgtaaat atttatgaac atcctcatct ttttgaggga agggattatagatcattcta 13680 attccatttt ctagcatttg gtaccattct aagcacatga taggcacccatttggagcat 13740 ttttggcttg acagaatatg catttagaat tgttcaaatt agaggtgtcagtgatgggaa 13800 ttagaatact atataattct aagtcatttg acttaaatac aaaagaatgattttccttgg 13860 tggggaatgg tgaagggagg caggagttaa gaagaggaga agagatcctaagtcatttat 13920 aaacttctct ggaaagacag gtgtgtgaag actttttaaa aagtcattcaccaaattgtg 13980 tgtgtgtgtg tgtgtgtgtt ttaaatagac tttatttttt agagcagttttaggttcaca 14040 gcaaaattga atgcaaggac agagatttcc cataaacccc ctgcccacacacatgcatag 14100 cctccctcat tatcaacatc cccaccagag aggtgtttgt tctagttgatgaacctacac 14160 tgacacatca ttatcaccca aagtccatag ttcacggcag ggttcactgtcggtgtacat 14220 tctatgggtt tgagcaaatg tataatgaca tgtatccacc attatagtaacatacagagt 14280 attttcagtg ccctgcaaat cccctgttct ccacctattc atccctccctctctgcattt 14340 ccacccccag cccctggtaa ccgctgatct ttttactgtc ccatagtttcggacgatcta 14400 tttttcagac agacacagag ctgtctttcc cttagtttct attctatcatttctttctcc 14460 ccatccatca taaaaggcta tgagtttttt ttaagtgttg aacaccatcctacttgtcaa 14520 gttaaaacat aagctcctgg ctgggtacag tggctcatgc ctgtaatctcagcattttgg 14580 gaggctgtgg cagaagcatc acttgaagcc agaagtttga gaccagcctgggcaacatag 14640 caagacccca tccctccaca cacaaacaca cacacacaca cacacacacacacacacaca 14700 cacacacaca cacaaaaaca agctcttgcc agaattagag ctacaaattgccctcaggtt 14760 cctagaagat cagtccttca attagattca gattgagatg cttcctcttttaaacaatga 14820 ttccctttct atcatgccca ataagaaaac aaataaaaat taaacaatactgcctgtaat 14880 ctcagctacc caggaggcag aagcagaact gcttcaaccc ggcaagcagaagttgcagtg 14940 aagtgagatc gcgccactgc actccagcct gggaaacaga gcaagattctgtctcaaaaa 15000 caaaacaatg tgatttcctc ctctaagtcc tgcacaggga aatgttaagaaataggtcca 15060 ccaggaaaga aggaagtaag aatgtttgac tagattgtct tggaaaaaatagttatactt 15120 tcttgcttgt cttcctaaca gttctccaaa gcttcgtacc ttggccagaggcttgtctcc 15180 tgcgtacctg aggtttggtg gcaccaagac agacttccta attttcgatcccaagaagga 15240 atcaaccttt gaagagagaa gttactggca atctcaagtc aaccagggtgaaaattttta 15300 aagattcact ctatatttta attaacgtca gtccgtcatg agaatgctttgagaaaactg 15360 ttatttctca cacctaacaa ttaatgagat taacttcctc tcccctcatctgacctgtgg 15420 aggaatctga acaagaggag gaggcagtgg gcaggtttcc ttatcatgatgtttgtcatg 15480 ttcagtgtga ggcctcacaa aaaaaaaaaa aaaaaaaaaa ggcgtcctggatataactga 15540 gagctcattg tacagtaaat attaataaaa cagtgattgt agctgaaggatagaactgct 15600 tggagggagc aagtgggtag aatcgcgtca aactaaagag catttctagccaaagacaca 15660 atgatagatt gaaggatatt tattctaaat atagaatatg ggtgaacgagatctgtggac 15720 ttctgggctc caacgttaga ttctgatttt agcaagcttg tcaggggattctgatattga 15780 aaggctgtgg ccttcacctg agaaacctgc cctagggggc catgaaaatttgtcctgtct 15840 ttcagaagtg ctatcagaca tcaaatggaa gttaaatcgt atcttaacaattactaggat 15900 gggcgcagtg actcacacct gtaatcccaa cactttggga ggctgaggcaggaggatcac 15960 ttgagcccag gagttcggga ccagcctggg caacatagag agacgttgtctctatttttt 16020 aataatttaa agagaaaaaa atactgaaaa tattgtatac accactgaattataataatg 16080 tgtatataat gtatatattc attatgagga atatttgatt atttcatatattatatcttt 16140 tccttctgtt tattttatcc agttatgaag tatttagaac aattcatcagtaattggggc 16200 taaattgaca gaatagtaat cagagaaaat agaaaaagac agatgggttatctttgaata 16260 ccaggttgga gttgtttatg ggtttgtttt ttgttttggg ggcgtttttttagacagagt 16320 cccactctgt tgcccaggct ggagtgcagt ggcacaagca tggcccactgcatccttgac 16380 ctcttgggct caagcaatct tcccacctta gcctcctgag tagctgggaccacaggtgca 16440 tgtcaccaca cccagctaat ttttttattt tttgtagaga cagtctttctatgttatcca 16500 ggctgatctc aaactcctgc actcaagtga tccccctgcc ttggcgtcccaaagtattgg 16560 gattataggc atagccacca cacccaacct agtttctatt tagacttggccctttcccac 16620 cagtcatttg tgtccaaaag atctcataaa tgtagacagg aaactgtcctttgctcatca 16680 gttttcttca tcctgtgtct agggggatgg tcggtggggg aaactggggttatgcaagtt 16740 cctctgaaac atcctctgtg agcccaggga tggatgaggc accagccgccagcgagtcag 16800 tgtgcagctt tccagaaagg aagtcatcag ccagtcagcc ggccctggcagccagcaccc 16860 ggcaaccctg ctgtcttgtg ataaagaaat ggtctgcctg acaggatggtgtggattttt 16920 cttttttctt tttttttttt ttgagacagg gtctggctct gtcgcccaggctggagtgca 16980 atggcgggat cttggctcac tgcagcctct gcctcccagg ctcaaggcatcctcccacct 17040 cggtctcccg agtagctggg accacaggca cacaccacca cgcccaactaagttttcgta 17100 tttttagtag aggcagggtt ttactatgtt gtccaggcta gtctcaaactcctgagctca 17160 agctatccat ctgccttggc ctcccaaaga gctggaatta caagcgtgagccactgtgcc 17220 tgaccagggt ggattttttc aagtgcacat gttgtggtcc cagaagctctgatggtacca 17280 aattccaagc gaaaaaaagt caatggttcc cacccatcct acctcccatgatggcaagag 17340 gaaatcacca cactgcagat acagtccatg taaaacaaat tgctatggattttgaaagtg 17400 aaccttaaga gaactgcact atgttttctt cattagagtt ctctggtaatttccagcttt 17460 tttttttttt ttttttagac agtgtctcgc tttgtcgccc agtgtcacccaggctggagt 17520 gcagtgacgt gatctcggct cactgcaacc tccgcctcgt gggttgaagtgattctcctg 17580 cctcagcctc ctgagtagct gtattttagt agagacgagg tttcaccatttggccaggct 17640 ggtctcgaac tcctgacctc aagtgattcg cccatctcag cctcccaaagtgctgggatt 17700 acaggtgtga gccactgcac ccggccagta atttcaagct tctgaggagccctttgaatt 17760 gttaaataac ttgtagctat gtccaacata tccatgttca gtgtatgttcgatatttctt 17820 aggaaacctg cccttggttg ttttctttgt ggtaattcat gagccggcaaatttgacatg 17880 tgttacagaa tatacctttt ctctgctctc ctacctcata accagaacttaattatcctg 17940 ctttagtcac ataaatagct aactaaataa atatatgaga tttcagtctgctcactgtga 18000 aaatagacct tctaaatgat ctcttccact tgcagatatt tgcaaatatggatccatccc 18060 tcctgatgtg gaggagaagt tacggttgga atggccctac caggagcaattgctactccg 18120 agaacactac cagaaaaagt tcaagaacag cacctactca agtaagaaatgaaaggcacc 18180 ctagagatgt tccagcccca aagatatttg aataggttgg actcgggcaccaatctagca 18240 agtcctacgg aagttgtata aagctgaaaa tactgaagca tttcccaaatgggaaatcct 18300 aaactcaaaa cttgcttttt ggtttttttg tttgtttgtt ttttcttcatctgacattgc 18360 ttagtagtca cagaatgaaa gataaatcaa tcattcatga tctaacaatgaccttcagtg 18420 ctctaaaaaa ctacggagtc aaggaaaaca tgaatatatt cctcatgtaaaattaaaata 18480 cagacatata aagggcaaaa catgaacatc attcatacct tgaggtccgtccccctccca 18540 gaaataaccc ccagtatgcc ttggtttaga gcattaagca ggagggccctgagtcactcc 18600 agacagtctt gaccaccaag cagcattctc tttttgtttc ctctgtggcttttgcaaaca 18660 cagggctagc tcagctaccc attagtatgt tttcagtcac taaaacagtcttccagtctt 18720 caaattagga tgacattgtc acatggggct ttaaagcaag tgaaacaaggaacccccttt 18780 tttttttttt ttgagatgga atctcactct tgtcgcccag cctggagtgcaatggcgcaa 18840 tcttggctca ctgcaacctc cacctcccag gttcaagaga ttctcctgccttagcctcct 18900 attcattatg aggaatattt gattattcag ttcctgtagg gtaaagatattacccccgat 18960 catattattg attattgagt agctgagatt acaggtgcct gccaccacgaccggctaatt 19020 ttttgtattt tttagtagag acagggtttc accatgttgg ccaggctccaggctcgtctc 19080 gaactcctga cctcaggtga tccacccacc tcagcctccc aaagttctgggattacaggc 19140 gtgagccacc actcctggcc acaatccttt tttaactatg aaatatatttttatctgaag 19200 tttgatgttt atacccaact gagggatgat gttcccatat ctcagttaaagaaataacct 19260 gctcagatac ttcaagctct tcttttgact tttgaaaata aatgatcttgaagttactat 19320 actttgtttg ggttagttaa cattatttaa agtatattat tttaattaattatctttgta 19380 agattttact gtatactacc tggagttcaa tgtatcagat ggatttcaaatttatgtaca 19440 ttttttatgt atatggtaca gaaaaaaatg tgatccataa gaaatcagaaaatagcgcat 19500 atgctaatag ctaatgttgt cctctaaaaa acttattttt gcatttttaagagggggata 19560 tactctgaca ctttaataag tgtaattaat tattgactgg aatttggcatgaggcagggc 19620 catttcagat cccattaaag gaatgacaca taccagagaa ccacagaagtaaggccacat 19680 ttgtaataaa tcattatagc tctgctagga gaagacccag ttgtattaggtaattaatgg 19740 atttgctctt aaaacacatg tcccggaaga tataggtgag tcttggggggccgcattaaa 19800 cattatacca atgtatctta catttctaag aaagttttac tactttacaggatctttctg 19860 ttaccaaaat ggaaggtttc caactccagg acttggcttt catagttcctacaccagggg 19920 aaatgccttc ctttgctaac tatgcaacca ggttagttag tgtaagtccagccaccctgt 19980 tggcaatgct aaaaggtaca acaaacacag aattttattt gcatttgtaaacatttgatt 20040 tctggctcga aattttcagt tttcatgggc acgtcatgga aacagaaatcttctgtgttt 20100 agtttgggca cctactcatt gtagtgacaa atatttcaga agccaataggggattccaca 20160 aattgttctg aacctgtggc tgagactggt aatggctgag tgacatggggacataccaca 20220 aaagaagagg tagcaaaagg ctgctgagat aaggacatgt tcattgcttagctagtggcc 20280 tgcaccctta aaacacatgt cccaggctgg gtgctgtggc tcacgcctgtaatcccagca 20340 ctttgggagg ctgaggcggg tggattacct gaggtcagga gttcgagaccaacctggcca 20400 acatagtgaa acctcatttc tactaaaaat acaaaaatta gccaggcatggtggcgggcg 20460 cctgtagtcc cagctactca ggaggcaggc aggagaatta cttgaatctgggaggcagag 20520 gttgtggtga gccgagattg cgccaccgca cgctagcctg ggcgacaaagtgagactctg 20580 tctcaaaaaa acaaaaacaa aaaacaaaca aacaaaaaac aacaacaacaaaaaaacggg 20640 tatcccagaa gatacaggta agttttctaa cacaggtcct cttgtatggtgcgttccact 20700 taagtagaag atgacaaaaa catttgtcat gagaatatag actcacattttaaacctgtt 20760 tgagcaggaa aaggaagcaa tgttacagat gtaattctgg gtgtgactgcagaaaggatg 20820 actcccttat taaagtagtc atcctgagtg agctaactct ttgtacttcctcttctcctc 20880 ctgttcccct catcacccca ttcttccgtt gcctacaccc aggcccacattggatgctga 20940 catagactta catggtacag tccaagggaa agatctgcca tttttttcaatgtgtcatct 21000 tggttatctt cattccaagg atctctccac tctttataca gtaagagatgagagtctgga 21060 aaggattggg aataagataa tgaattgtaa gttttaaatt gttcttcgtattttggggaa 21120 ggagtaggct aggtggtcct tctgtttttt ttttgttttt ttttttaaagtagatgtggc 21180 cagacgtggt ggctcacgcc tgtaatccca gcactttgag aggctgaggcaggtggatca 21240 cttgatgtca ggagttcaag accagcctgg ccaacacagt gaaaccccgtctttactaaa 21300 aatacaaaaa ctagccgggc ttggtggcgt ccacctgtag tcccagctactgcagaggtg 21360 gaggcaggag aatcacttga acccgggagg tggaggttgc agtgagccaagatcatgcca 21420 ttgtactcca gcctgggcga cagaacaata ctctgtctca aaaaaaaagagaaaagaaaa 21480 gaaaaaaaga atggatttga actcagtcgt caatagcctc tattccaggagatgttacag 21540 ttgattatgt tatagggggt gtataataga atttcgagct atgtaaattccaagtgcatt 21600 tggaagaatg aagaaatgga ggaagggtaa agtatgagtg caagcattccaggttttttg 21660 aaaatgctat aatctttgtt cagggctagt acaaagtgct atttagctgtaagggttttt 21720 tgtgatttac agacagtttt cacatgtgtc atttcaacct tggttttatggcgaaggcat 21780 gtgatggtgc ttgtcccagg actttagatc catatctgag gttcctgtcgggcaaagata 21840 ttacccctga tcatattata gtctataagt gggagagttg tgcctggagctcaagtctta 21900 tgatttctga tccagggcac ttcctacaac atgattttgc aatataaaagcctataatgt 21960 gtgactaaag caggtcactc accccttgta acagactcta gtaatggtactgccaccaaa 22020 cggctgcgtg atattgggca aagacttacc ttatttgaat ctcagtttcctcctagaaaa 22080 atgagggtgg aggttaagca taggctgatg atcctaaagc ctccatactgccctaaactg 22140 tggctctaag atccagtaga atgctgggtc acaggactct agggagcttttcaaacccaa 22200 atgtctgtca ttccttgatg gtaggcagca gtttatggaa gtgggcgacacagcaaatat 22260 caaaatacct aaagcagctt gcaagagttg tttctgccta gtggtctttatagttaatat 22320 taaatagtta attttttttt tttttgagac agagtcttgc tctgttacccaggctgcagt 22380 gcagtggcac aatctcggct cactgcaacc tccacctccc gggtttgagcaattctgtct 22440 cagcctccca agtagctggg actacaggtg catgccactg cacccagctaatttttgtat 22500 ttttagtaga gacggggttt caccatattg ggcaggctgg tctcgaactcttgacctcag 22560 gtgatccacc tgcctcagcc tcccaaagtg ctgggattac aggcatgagccactgcaccc 22620 agcttaaata gctaatattt aatattattc tatagttatt caagtaattcaggccaaaga 22680 cttagaaaca aaacaaaaag ccacttttaa ggagaaaggg tgtaagtttgccagatagat 22740 agagatcttt cttttttaac tacaagagtt caggaatgaa ttactctttaacaaacgact 22800 atagatatac atgaaaattg gaaggactta ttatgcatat gataatcaatttaaagacaa 22860 cacttaaaat tatattgttg ccactctcaa aaagtggtaa tagaacagctaatggtttaa 22920 aaagcagagt acagaagttc ccaaacttat ggcaccttaa tatcgcagaaaactttttaa 22980 agcatgccta ggccacaaaa aatacctgta ttttgattat taaattgtaaggtctacaca 23040 acctaatagt aataggtcca atagtaatgc tgtccaatag atgttgatgtttttttcctt 23100 gcaaacttaa aagatcctac agtgcctctg taaatagcac tgcctggttagagttgaatt 23160 tcagataaat aatttttttc atgttaatta tttttctttt ctttacttttttttttgttt 23220 ttttgttttt ttgttttttt ttttgagaca gggtctcatt ctgttgcccaggctgctgtg 23280 caatggcatg atcatggctc actgcagcct tgacctccct gggctcaggtgatcctccca 23340 cctcagcctc ccaagtagct agctgggact acaggtgctt accatcatgcccggctaatt 23400 tttgtgtttt ttgtagagat gtggttttgc catgttgccc aggctggtcttgaactcctg 23460 ggctcaagtg atccgcccgc ctcggcctcc caaagtgcta ggatgacaggcatgagccac 23520 tgcacctggc ccctgggcga agtatttctt aatggttaca taggacatacactaaacatt 23580 atttattgtc tatatgaagt tcaagtttaa ctaggtgccc tgcacttttagttgctaaat 23640 cctgtagctg tacccatgca ttcactggtg ctccccagct tgccttgcacagagtttgga 23700 aaccatagtc ctataactct aggccaattt tttaatgtaa aatttgattcattttaaatt 23760 aataaataat aacaggaatt tttttaaaaa ttgttttaaa tataattaaaattatcaaaa 23820 tattttttaa ctgaacttgt gactagagat atttagatta tgaagagtggggtttatgct 23880 aactaatgac agtctggcta tgcatgtgga gcactgagct ataaattgtggcttccccaa 23940 ttctcctgat gtcacttgaa caaaacctaa gtgtcagacc agagcttctggtatcttcca 24000 tgggatttca ttcaacagct ggagcaaatg aagtcagatt gattttttttaatttgtcca 24060 attttgttgt ctcaaaaaca taattataat catttattag aactagaatttcttcagttt 24120 aacaacagaa atagttattc attatgaaaa gcgaatctgg aggccttcattgtggtgcca 24180 atctaaccat taaattgtga cgtttttctt ttaggaagct ctgtagatgtgctatacact 24240 tttgcaaact gctcaggact ggacttgatc tttggcctaa atgcgttattaagaacagca 24300 gatttgcagt ggaacagttc taatgctcag ttgctcctgg actactgctcttccaagggg 24360 tataacattt cttgggaact aggcaatggt gagtacccca gggaacaattcattaataag 24420 gagattcccc actagcatta tttcttttct tttctttttc ttttcttttttttttttttt 24480 gagacagagt ctcgcactgc tgcccaggct ggagtgcagt ggcgccacctcggctcactt 24540 gaagctctgc ctcccaaaac gccattctcc tgcctcagcc tcccgagtagctgggactac 24600 aggcacccgc caccgcgccc ggctaatttt tttttttttt tttttttttttttttttgca 24660 tttttagtag agacggggtt tcaccgtgtt agccaggatg gtcttgatctcctgacctcg 24720 tgatctgccc tcctcggcct cccaaagtgc tgggattaca ggcgtgagccaccaggcccg 24780 gctagcatta tttcttatga cacttttttt ttttttttga gacggagtctcgctctgtcg 24840 cccaggctgg agtgcagtgg cgccatctcg gctcactgca agctccacctcccaggttca 24900 cgccattctc ctgcctcagc ctcccgagta gctgggacta cacgcacccgccaccacgcc 24960 cggctaattt ttttgtattt ttagtagaga cggggtttca ccgtgttagccaggatggtc 25020 tctatatcct gaccccatga tctgcccgcc tcggcctccc aaagtggtgggattacaggc 25080 gtgagccact gcgcccggcc aacactcttt ttattattag caaatatacttctgcctggg 25140 cacattcttg caagtgctca acaatgcaac ttttggaagt gcatgtggcagaaactcctg 25200 ctgtatttat tccagaacct attattgcta atcccagttt atgttacatttgaagtgaga 25260 accagttgga gccagcaacg ttcccagctc caaagttccc ttgagattttcagaatcact 25320 taaccctatt atgcttggca acctggactc agcaaaactg ggaagtcagcagtttgtttt 25380 attcatccct tcctttctca gtttctcaaa tgtgtcagtt aatctcagtaaccccattgc 25440 aaccttcatt acctgcccaa gcggtctaga acttgccagt atagaatcctacgtgggtca 25500 agctcctgac tgtctccttc ttcactcttt ttttgcaaag aacttgtaaattttaactat 25560 aagtattcat gattcgccac atttattcaa aacatagagt gctttttccacatatcagcc 25620 aatggaaata aggattaaat gggaaatgaa atgtagtaat aggataagcacaagtcttct 25680 tcctgctcaa actttttttt tttttttttt cagacaagat cttgctctgttacccaggct 25740 ggagtgcagt ggcgtgttca tagctcaatg taacctccaa ctcctgggctcatgcaatct 25800 ctcacacctc agccccctga ttagctagga ctacactatg cctagccaattttttttctt 25860 ttgtctggtt gtgttgccca ggctgtctcg atctcctggc ctcaagtaatcctcctgcct 25920 cggccttcta aagtgctggg attataggca tgagccactg tgcccggtctcaaacctttt 25980 tttccaaagt aaatgaagtt attagatatg gaatatagtc tagttcccagatatccatat 26040 ccattggttt attaccctca ttattaactt caaattgttt aatagaccctcatatctcag 26100 ttatacagtt aaaatttttg ttttgttttt ctggagtatc ttatttataactatgagttt 26160 tactttactt atttatttta ttttttgaga cagacgcttg ctctgtcactcaggctggag 26220 tgcggttgcg tgatcatggc tcactatggc ctcgaccttc tgggctcaagtgatcctctc 26280 cctcagcctc ccaagctgag actacaggca tgcaccacca catctagctaattttttttt 26340 ttccccatgg aacaaggctt tactatgtta cccagagtgg tctcaaactcctggcctcag 26400 gggatcctcc tgtctcagcc taccaaaatg ctgggattac aggcatgagccatagcgcca 26460 gacctggttt tacttttctt gactttgaat tacaagtttt tgtaatttggaaaatgtttt 26520 gttgctttta aatactgctg tatgtttgct tttaaataca acatttctcgatatatattt 26580 tgagaattgc tgtctttcag aacctaacag tttccttaag aaggctgatattttcatcaa 26640 tgggtcgcag ttaggagaag attttattca attgcataaa cttctaagaaagtccacctt 26700 caaaaatgca aaactctatg gtcctgatgt tggtcagcct cgaagaaagacggctaagat 26760 gctgaagagg taggaactag aggatgcaga atcactttac ttttcttctttttccttttg 26820 agacagagtc tcactctgtc agccagactg gagtgcagtg gtacaatcatggctcactgc 26880 aacttcgacc tcccaggctc aagcaatcct cccatctcag tcccacaaatagctgggact 26940 acaggtgcac atcaccacac ctggctactt taaaaaaatt tttttgtagagatggggtct 27000 ccctgtgttg cccaggctgg tctcttgaat tcctgtgctc aagccatccttccacctcag 27060 cctcccagag tgccaggatt acaggcatga gccaccacac ccagccaccacttttcttaa 27120 aaaaaaaaaa agattctctc tggtagacaa tcctcaatag tccacatgttattaaacaat 27180 ctgctgcctg aatacatgat ttaccaaaaa aaggaaattt tgacgggttcagaatatcaa 27240 gggatctgag gcaaatgtca cctatgataa aatttgctat caaaattaggaagtttgtgt 27300 ttacctgatc ctaaagcagt aaccagccca tttctaggga ataaaactctcatgcgtata 27360 ttgtgcatat atatgtatta tatgactgag tgataataaa attttttttctagcttcctg 27420 aaggctggtg gagaagtgat tgattcagtt acatggcatc agtaagtatgtctcctattc 27480 ttaatactag gaaagtaagg ctagctttat ttattaccta gtattcaaaaagttagttca 27540 tttaactgcc aattgactgc agttcaaata agaaacaaat agtgtctcaagtagcactgt 27600 actccaattt taatattaat aaaaaaaatt ttaagttatt ttaaataatgtagtggtttc 27660 tataaagatc actttataca gaagaacagt gccaattaac ccatggaacatataagtagc 27720 taaaaccaat tgcttgccaa agaaccagta acccaggagt acatgtccttgccactgtgt 27780 tttttcaaga cagagtaact gatttctagt tacttgcata gaatggactcctcctcataa 27840 ctcccttcca tcttggtctt tccctagtag aacttctacc tttttttagtaacaggtgag 27900 tgggagaggt aagaaggaga ataaggtcag caattaacct aaaagcagaaagtaaaattt 27960 gttatttttt ttctgaatat tttctgtgta atttagctac tatttgaatggacggactgc 28020 taccagggaa gattttctaa accctgatgt attggacatt tttatttcatctgtgcaaaa 28080 agttttccag gtaatagtct ttttaaactt tttaatgtaa aaccagaatccttattttat 28140 agtctagcta gttctaaatt ctataggtat gtatatttac atgtttttctaattttagag 28200 aacaagcact atgacttatc cactgttagt tttcccctta gcattgggtcttaccccatg 28260 tacgtgatta gaaatttgaa atatttccaa tagcctttag tagaattaactcacatagat 28320 gataagaatg ggttggttca cttcatgttc cttccacagc ctactatttcaataaaagaa 28380 agtttcccaa gacctaaatg actatgaaca tattttataa ctatataggaggggtgggtc 28440 taggaataca aagttttgaa tgctgttaat cttcaacacc acagttgaaaccacaggtca 28500 gcttttttgc aattaccatg gatacttttc tgttctatag gtggttgagagcaccaggcc 28560 tggcaagaag gtctggttag gagaaacaag ctctgcatat ggaggcggagcgcccttgct 28620 atccgacacc tttgcagctg gctttatgtg agtgaagcag cgctggccttaggggtcaga 28680 gtgcagctct tctccatcct tctattctgc tgaaatagct ccccagccaaaaagcagatc 28740 aaagaccgtt tcagtggctg agccccaaaa ttcatgccag attttgcaagaaaatgattt 28800 actaaagctt gagggacatc tttaacaagt gttccaaatt aatcactataaggatgaatt 28860 gtttcagaaa ttttggcctt taattatggc ccataaatat gtcaagtagtccttactcta 28920 aagaagtaca ctgtaaaaga atgcatatag ccggatatgg tagttccctgtaatcccaat 28980 actttgggag gccaaggtgg gaggattgct tgagcccagg agtttgaggctgcagtgagt 29040 tatgatggtg ccactgcact ctagactggg caacagagtg agactgtctttttttttccc 29100 ctctgtcacc cagactggag ggcagtggca cgatctcacc tcactgcaacctctgcctcc 29160 cggattgaag cgattctcct gcctcagcgt cctgagtagc tgggactacaggagtatcac 29220 cgcactgggc taatttttgt atttttagta gagacggggt tttgacatgttgcccaggct 29280 ggtctgaaac ccatgagctc aagtgatctg cctacctcag ccttccaaaatgctgggatt 29340 acggacatga gctaccacgc ccggccacac cctgtctctt aaaaaaaaaaaaaatgcaag 29400 ttagagcata ttacagcttt gtctctcagg aggatactta gtgtatgtagctataattca 29460 tagattccca agaagtttag agcctaaagt atgaggtccc accagaggggctatcattaa 29520 atttaaagat ttgttaaatc atctcattgt ccaacaccac aaacttgattgctttaaaat 29580 actggtttag ttacatttag taactctatt agtgctttta atctatactgctatatcctc 29640 acattgagat tttttttctt ttctcttcca tcttcattct tttttctctcatcctcattc 29700 ttataagcct agaatacatc acaaatcctt tatgcccatg gaagcaagaggaataaagaa 29760 tggagatgtt tgttttgcca ttaactaaag atctggggtg tcggggagaagggggataga 29820 gaaggagaag tgggaagagg tgtccataat agcttaggtg caattctgcttattttacat 29880 tttacccccg ctgactgcca ctttttcttc agccctcaca cattgtttgtgcagggacct 29940 cataggacca ggaattgtct atagaggtgg gaatttgtct caccctgaaagggatacctc 30000 tagcatggta atagtcttct aggatttgtt atcatatgga aagatgtaaagggagggatt 30060 ctgctgctgc tgctgctgct gcatgcagtt gccatttcat ttaaatgacttatttataat 30120 tgatgacact tttctggctt cctgttaatt cctccctcaa agatcaataaaccagaacca 30180 ggcatggtgg catgcacttg tggtcctgta accacccaac aggttcaccttgcctgctgt 30240 ctagatagag ccaattatca agacagggga attgcaaagg agaaagagtaatttatgcag 30300 agccagctgt gcaggagacc agagttttat tattactcaa atcagtctccccgaacattc 30360 gaggatcaga gcttttaagg ataatttggc cggtaggggc ttaggaagtggagagtgctg 30420 gttggtcagg ttggagatgg aatcacaggg agtggaagtg aggttttcttgctgtcttct 30480 gttcctggat gggatggcag aactggttgg gccagattac cggtctgggtggtctcaaat 30540 gatccaccca gttcagggtc tgcaagatat ctcaagcact gatcttaggttttacaacag 30600 tgatgttatc cccaggaaca atttggggag gttcagactc ttggagccagaggctgcatt 30660 atccctaaac cgtaatctct aatgttgtag ctaatttgtt agtcctgcaaaggtagactt 30720 gtccccaggc aagaaggggg tcttttcaga aaagggctat tatcatttttgtttcagagt 30780 caaaccatga actgaatttc ttcccaaagt tagttcagcc tacacccaggaatgaagaag 30840 gacagcttaa aggttagaag caagatggag tcaatgaggt ctgatctctttcactgtcat 30900 aatttcctca gttataattt ttgcaaaggc ggtttcagtc ccagctacttgggaggctga 30960 gacaggagga ttaatggagc ccaggagttt gaggttgcag agagctatgatcacgccact 31020 gcactccagc ctgggtgaca gagtgagacc ctgtctctaa ataaataaataagtaaataa 31080 ataaatacat aaataaaatc aagatggtgt gcaattagaa ttgagcgattttgtttccaa 31140 acctcaagaa agcttggtct tgctctgtcc caggtggctg gataaattgggcctgtcagc 31200 ccgaatggga atagaagtgg tgatgaggca agtattcttt ggagcaggaaactaccattt 31260 agtggatgaa aacttcgatc ctttacctgt aagtgaccat tattttcctaattctagtgg 31320 agtagattaa agtcaactca ggacctctgg tgttaacctc ctatgaacagtcagtcctct 31380 cagtaactag ccaaatcatg agatgatgaa ttagaaggag ccttagatagcatccaatct 31440 aacatttttt tgtgtgtttg aagagaagaa atcaagagct aggaataactttttaaaggt 31500 aagccatttg cagtatagtg tggattttgt ttaaaagggg ataatttgaaattttatgac 31560 tcattataca agacaaaata agttggattt tcaaatgttt tacaaagtaaatcaaagtta 31620 taattgccta cagtacgcaa agcttcaaaa cattttttat gttatgaaattgtaatttat 31680 ttaaccttaa aatgagccag taccatgtgt ttgcttaaaa atctcatgctaagaatttac 31740 tatgttgtta ataatcttca agatatttat gaataaagtc ttatttctaatccttcctcc 31800 aactgtatct ggtgctaaat caggaaatgt ttcttcccaa aaagcctcgtggaagatctg 31860 tatgtctaaa tatatgtcag ggataataca gatgtagccc tgcgaagcatgaccttgatt 31920 tttatagtct aaaatgtcat ttgcagatat ctattttcta agaataattcctaaaagaat 31980 tatttgaatg ttgtaggaaa gctaagaaat tttgcaaaga gcgtacgtgaaaatataagc 32040 taggcttttg tggtttgtgg atagacttcc caacaaaatt gctttttatctatagtgatc 32100 caagcttgtg gaacatatta gtcatctttt tttagaaaat tcttagaaaagtgatcttgc 32160 aaaaatggaa tttatctttc cccaagtata ttctgtcatg tatagagttaaactaagcat 32220 agtaatttca ccagacaaac attcaaaatc tactcctgac ctttttatctcatccaaatt 32280 ttcccagggc ccagacataa acctttgcct tacgaactct ttgtatatgcactaaatatg 32340 cttctccttc aaggttctca gtcagctaga aaaatgtgca agagtaaatggtacccttct 32400 cacttgtaga tccaagagaa ttagacttaa actcactcta catgtctgtgactttatttt 32460 atttgcatga cagtcctgtg aggtggcaag gcaggtatct tggatccattttttagataa 32520 ggaagttcaa attgagaaga ggttgcatga tttacaggaa gccatactgtagtcctatgt 32580 tactcttaaa aatcccattc aaatcctgct tctgaggcct gcatactttctaccctacca 32640 gtcattgacc catgcttatg tctcctttga aaacattgat tccactcttgtctccagtga 32700 aaaagtggaa tttaagcaga gaaacaaaag ccatttgtct tgttaagtctactttccctc 32760 tactttcaag aaggaaagtt ggggtatgtg ttgaatggtg atttatttatttatttatta 32820 ttttaaaaat tgatacaagg tcttactgta ttgtgcaggc tggtctcaaactcctgggct 32880 caagtgatca tcccacctca gcctcccagt gttgggatta cagcatgaaccattgtgccc 32940 accaccgatc cgcagttttt taagaaaaac ttttactata gaaaattttaatcatataca 33000 aaatacagag gaaagtatat gaacccactt taggagacta gaatatgccaccccaaaata 33060 tgccactttg gcataaggat tatttcgagc taaaggcaac tgggaagaaacacatagaag 33120 aaaagttctc tgtccttctc catttgccta aaagcaggac atgaatcttaaaagtccccc 33180 tccttccctt tctaccagga aaaacaagag ttaatcactg aagataacttcagaccctta 33240 tcagtgtaga gatggcacta gaagaatcta tattacatac tcatttattttccttcccac 33300 aacttgccac cccagagact aaaaatcctt ttcctttgtc atgtctcttgtccaaaaatt 33360 tgctctataa gctggagttc taagccacct ctttgagaat tacttgttccctggtatttt 33420 ctgttaacat acatgtatta atatacatgt taacaagctt ctgtttgtttttctcctgtt 33480 ttctgtcttg ttacagaggt ccatcccaac taagaactaa agagtaggaggaaaatataa 33540 tttcctcctg catactttga tcttgtttaa tccgtaaccc ttcccacttttcacctccta 33600 cctattagat tactttgaag caaatttcag atatattact ttatctataaatatttcagt 33660 atgtgctagg tgtggtggct cacacctgta atcccaacac tttgggaagctgaggcagga 33720 ggatcacttg agcccaggag ttcaagacca gctacggcaa caaaaaatcaaaaacttatc 33780 tgggcatggt ggcacatgcc tgtggtccca gctacatgag aggctgaggcaggaggatcg 33840 ctttagccca ggaggttgag gctgcagtaa gctgcattca caccactgcactccagcctg 33900 ggtgacagag taagaccatg tctcaaaaaa atacatattt tagtatgtatcctttttgta 33960 aaaacacaat acttttatca tactttaaat aataacaata attccttagtatcaccaaat 34020 attttgtcag tgtctcacat tttccttatt gtctaaaata ttgttgatagttattcaaat 34080 cagaatccaa acaaggtcca tatattacat ttggttgaca agtctcttaagtttgttcat 34140 ctttaagttc ttcctccctc tctttcatct cttgtaattt attaatgtgaaaaaacaggt 34200 aatttgttct atagtatttc ctacattata gagtttgcta catttattccctatgatatc 34260 atttagcatg ttcctctgtc ccctgtgttt cctgtaaact ggtagttatacctagaagct 34320 tgagtttatt caggttttta attgtatttt ttttgcaaga attctttattatctgcttct 34380 ggaagcacag aatgtctggt tgtgtctggt tttgatcttg acagctactgatgaccattg 34440 cctaatccat tactttattg gggtgggggg aataaggttt taaaataaattttttttaaa 34500 gattttttta actgttattt tgagacagtg tctcatttcg tttcccaggctggagtgcag 34560 tggcacaatc acggctcact gcagccttga cctcctggga tcaggtgatcttctcacctc 34620 agcctcctgg gtacctggaa ctacaggtgc acaccaccac acctggctaattttttgtat 34680 tttgtgtaca gaaggggttt catcatgttt cccagactgg tcttgaactcctgggttcaa 34740 gtgatctacc cacttcagct tcccaaaatc ctgggattac actttggccaccgtgcctgg 34800 cctaaatgaa attatttgtc tctaaacaga cagaagtttt actttaaaaatttgtctttg 34860 tgtgtacatg tgtttgtgta tgtgtgtgtg tctaaaagtt tggctttgagctttgctttg 34920 aattcttgga tgaacaataa ccaagaatac ttaaactctg atcattcttgacagatatcc 34980 cctacaggct atggcctttt gaattgtgtc ctccagtgat aaaaagcagcaagcacgata 35040 ctgctctcag attcatggtg gtcacatgtg aggtgaaaaa aaaaaaaaagatgaatccta 35100 tttaaatgcc cccaggataa cagtgatact ctttgtagga taactatttgcttgccactg 35160 gtttcattaa ataaggacat aagtaaagat ctatttttgt ctctttctccccaaccacca 35220 caactaggat tattggctat ctcttctgtt caagaaattg gtgggcaccaaggtgttaat 35280 ggcaagcgtg caaggttcaa agagaaggaa gcttcgagta taccttcattgcacaaacac 35340 tgacaagtaa gtatgaaaca caccctttac caatcatcaa gttttagtgggtaagcctgt 35400 aactttactc aaacaccctg ttgcatgtgt ctatacattg cataagtataggcagttgca 35460 atttagtaaa gttttataca acgattttat tttattttat ttttagaagaaaaatgctac 35520 ttttgttgtt gttgtttttt gagacggggc ctcgctcgtc acccaggctggagtgcagtg 35580 gtgcaatctc agctcactgc aacctccgcc tcccgggttc aagtgattcttgaagaggag 35640 aacaataata acaacaatat tattttcaaa agttgtgacc gcagtttctggagttgagaa 35700 gacatcgaga tttttgtagc ctcatactct tgctttaggt agcaaaaaatgttcctaaat 35760 ctcaggaata ttctctagat aggtttcaat ctatcattcc tgataagatgatgctgaaat 35820 actaattcta gccaaaaaag accagctacc atttccgatt gttggggactgggaactctg 35880 gatagtgagg accccagtag gaagtagcga ggggaatggt ttgaatggataaattcataa 35940 aaaatgtcag tagatttaat tttcttatac atttcagtct ttttataaggctaggaaaag 36000 cccctgtttt tatggtttat aatttgaatt cacatgaacc cacaaaatttgccttttacc 36060 ttcctatgtc tgaaaatgga tagtctggct ggcctcttaa caacccagctggcagagctg 36120 tgaggatctc agtgtgctct agcccagaca ttggtagcat gaacggcaacatttttaatt 36180 gtgttttcaa aataggagca cactagcggt ctaaaacgat cataaaagaaggatactaag 36240 agggcccact gtcattatgg atcctaatac ttaggatgca ttatggattgtcattatgga 36300 tactaatact taggatcaca tttgtaattg agtttttaat tgcttaaattagatacatat 36360 ttctattaag ttaacctctt tgcttttagt ccaaggtata aagaaggagatttaactctg 36420 tatgccataa acctccataa tgtcaccaag tacttgcggt taccctatcctttttctaac 36480 aagcaagtgg ataaatacct tctaagacct ttgggacctc atggattactttccaagtaa 36540 gtaattttcc ttgttcattc caaactttca ataaatttat tggtgtttatcagaatagag 36600 agtttggaca gggagcaaaa gacaaagtca actatatcaa gttctaataattcttaatat 36660 tcaggaaatt tatgtatgaa tacttactaa tatgagtata actcatcctaagagtctaaa 36720 gcaaaaggat gtgaacacaa actagcagtt atcttagaga ataagtttgcatttcaaaat 36780 aacttgacat atcaagatcc actcaacgca tttaaattat ttactctaaaaagacataat 36840 tcttggtaac acattcacta aagcaaaata tacctttata taattgctatcaaaggtatg 36900 tgggttggta taaaatatca taccatgtga gatcagtgtg attcctttacagcattaatt 36960 tttattggtt agagtaagaa aaagaatagc tagagtatat ttcttaagtagattctcata 37020 cactttggtt tcaaaaacca attattgact acatcttata aaagcctgtattcaatggag 37080 tgccaaaaaa tgactatgag tcttaaagag ttaggcatat aaatattttaaggtttctgt 37140 tcaatgtatg ttggaaggag ttcctttctc atgactattc tcatattggagcataaaaag 37200 agtttacagg cttggcgcag tggctcatgc ctgtaatccc aatactttgggaagctgaag 37260 caggcagatc acttcagccc aggagtttga gaccagcctg ggcaatatggcaaaactctc 37320 tctacaaaat ataccaaaat tagccaggcg tggtggtgca tgcctgtagtcccagctact 37380 tgggaagctg aggtgggagg attgcttgag cccagggggg tcatggctgcagtgagctgt 37440 gatggtgcct ctgtcaccca gcctgggtga cagagtgaga ccctgtctcaaaaaaataaa 37500 taaataaaaa ttaagagttt acaaaattct caccatctcc tcccatctttgcaaatgcca 37560 cataagtgat gtgttccagg actattagcc tcggaacctg aggcagtacagtaagcacgc 37620 tttctccaaa gtcctgtccc ccacagacaa acattattta cactgggtactgctctttta 37680 ttttttcccc tctatgcttt attttactat aactataatc atataacatgtaataggaaa 37740 aaggcagggt cgggggagag atccagaagt cttcccaaga gcctttccaacatagcctct 37800 gtagacattt tttctttctt cttttttttt tttttttttt ttctgagacagagtctcact 37860 ctgttgtcca ggctagagtg cagtggcgtg atctaggctc actgcaacctccgcctcctg 37920 ggttcaagca attctcccac ctcagcctcc ctagtagctg ggattagaggcatgcatcac 37980 cacgcctggc taatttttgt atttttagta gagatgaggt ttcaccatgtgggccaggct 38040 ggtcttgaac tcctgacctc aagtgatcca cctgccttag cctcccaaagtgctaggatt 38100 acacgagtga gccaccgtgc cctgccccta ttacattctg atcacacatttcatgtttta 38160 taattggaaa actggtgaaa ttatagacaa tgttttgttc ccctaaattctctttgatga 38220 gtatatatta cttacactct tctgtcttta aaattttgca aaatagtatcctagataagt 38280 ttatgagtgc acagtctgta cgcttactca tattaatgac ctcggagagttaaacaacag 38340 tcacctttaa aaattattac tatcattatc attatttttg aggcgggggtctcattctgt 38400 ctcccaggct ggagagtagt ggtgcggtca cagctcactg cagccaccgctacctgggct 38460 caagtgatcc ttcctcctca gccttctgag tagctgagac cacaggcttatgctaccaca 38520 cctggctaat tttttaactt tttgtagaga cgatgtctca ttatgttgcccaggctggtc 38580 tcaaactcct aagctcaagt gatcttcctc agcctcccaa agtgctgggattacaggcat 38640 gaaaaactgc acccagccct aaaaattatt agggtcctgc atagtaagactttaataaat 38700 atttaaatga acatctggtt tttttaaaaa aaaaatagag acaaggtctcactatattgc 38760 ccaagctggt ctcgaactcc tggactcacg caatcctgct gccttagccgcccaaagtgc 38820 tgggattaca ggcatgaccc acctcatctg ggctgagtga acatatttttaacataaagg 38880 ccgtatttta tatttatctc atacattttg cccagcatcc ccatttccgccgaatctgtt 38940 gcttgctaat tccttccagc ttcatttcat ctgaaatttg acaaacatcttctatttctt 39000 tgtcgtcatg ttattgactt cagaatataa aataaaacac tatacccaaattaaacccca 39060 ccctcattgc ccagcctgat gtgaaaataa tcagcataca ttaagcttacccttgatata 39120 tgtgtagcat cttttagata aatatacagc tgattaagca atatagcctgatggtataat 39180 atcttgccca tgtacctcat cttatctcca gcaggattaa ttcacagtgatcagatttac 39240 ctttaaactt tgtagcaaaa tatcctctcc aaaagcatat ctaaaacttttgtgtgtact 39300 cttgcaagtt tcttaatttc atgcagaaca ggctcttacc actgttagctggagatattt 39360 tcaagaccta tttttgtttg tggtttcctg atgatggtca tggcatttcccccttcactc 39420 catctaaaaa ttgaggtgat acaggctttt aaacaaaacc aactcatatagactgagtac 39480 aactgcaatg caggcatgct aacctctgct acaatcatgg gcgtgctattgatatgtctt 39540 aagttacaga acacagggct gagcgtctca ttaggtcaaa atgtaaaccagtttttctgc 39600 tcactgatgc ttaatgagga cagggtgtga gagatttctt taaggaaaacaaatatataa 39660 taatgctaca tggaaaaata tctaacatta gagaattaag taaataaactaatatactca 39720 caccatggaa tcttgtgcag acattaaaat tatgtagtgg atggatgtttaatggtgtga 39780 gaaaaagtta ggatgtgctg gggtgggggg aagaatcaag ttttaagaaaatacagtata 39840 cccatactta agtaaaaaaa aaaaaaaagg tatgtacagt catgtgttgcttaatgatgg 39900 ggatacattc cgagaaatgt gtcgataggt gatttcatcc ttgtgtgaacatcatagagt 39960 gaacttacac aaacctagat ggtctagcct actatgtatc taggctatatgactagcctg 40020 ttgctcctag gctacaaacc tgtaaagcat gttactgtag cgaatatacaaatacttaac 40080 acaatggcaa gctatcattg tgttaagtag ttgtgtatct aaacatatctaaaacataga 40140 aaactaatgt gttgtgctac aatgttacaa tgactatgac attgctaggcaataggaatt 40200 ataattttat ccttttatgg aaccacactt atatatgcgg tccatggtggaccaaaacat 40260 ccttatgtgg catatgactg tatacatgta cacaaaaaat agatgaaagaatgaatatac 40320 atcaaaatat ttaaaatggt tataatgact taggttactt ttatttatcttagtaataat 40380 aatgatgata gataatactt ttatagtgtt tactatataa aagacactgttataagtgtt 40440 ctacatactt tacatgtatt acctaaatga tataaatata actctgacagtaactaatct 40500 tatacgttct cttttctttt tttttttttt ctttttttag acagaatcttgctctaccag 40560 gctggagtgc agggtgcaat ctcggctcac tgcaacctcc gcctcccaggttcaaacgat 40620 tctcatgtct cagcctcctg agtagctggg actacaggca cacaccaccatgcccggcta 40680 atttttgtat ttttgggtag agatggagtt ttgccatgtt ggccaggctgatcttgaact 40740 cctggcctca agtgatctgc ctgcctcagc ctcccaaagt gctgggattacaggtgtgaa 40800 ccactgtgct cggcctaatc ttacaagttt tcaatattta aagagtgctaactttgttga 40860 caatataaaa catatttgag aaaaagagat ataagcatct tatttagaattatgaaaata 40920 tcaatagacc tacagccgac taaagctttt cttcataagc tcttgcctatattgattcgc 40980 tcctgtgaat atgcattaat ttgatttaaa taataagtat gtataagaaataacactttt 41040 ccttaatttt taagaacgtt caacagtttt taatttgaat tccaatagtgaaatacatag 41100 aaaatataaa attttctgta gtttagccaa attgtttttg tttcaccacagcattctacc 41160 aaaatttctt aataacagta agaaaatgaa tgcatacctc ctgcagggagaggggagtta 41220 ggcagtttat gggcatagtt acaagtgaga aatttcattg gctaccatttacgctaaatt 41280 cataaaaact gcattcaatt ctatatatct attttcttta cataaaaaaggtttcaatta 41340 ttggccatta aataaaatag ccaccattcc agaagttgtg tcatgtttatcctttttata 41400 ccaccatcat attgcctatt atatagattg tgtgtgttcc attttctgtaatgggccaga 41460 cagtaagtat ttctggcttt ggagtccata tggtctctat cataactactcatctctgcc 41520 attgtagctt aaagattatc taggtcaaat gcctaagtga tatagtgttgaaatacaagt 41580 tatataatat aggctgccac aaaaaaaaat ttatttggtc taaaaaagatttcatgactt 41640 ttgtagcagc atgggtgggg catgcaccac ttggttaact cggtgtatctttctcctttg 41700 cagatctgtc caactcaatg gtctaactct aaagatggtg gatgatcaaaccttgccacc 41760 tttaatggaa aaacctctcc ggccaggaag ttcactgggc ttgccagctttctcatatag 41820 tttttttgtg ataagaaatg ccaaagttgc tgcttgcatc tgaaaataaaatatactagt 41880 cctgacactg aatttttcaa gtatactaag agtaaagcaa ctcaagttataggaaaggaa 41940 gcagatacct tgcaaagcaa ctagtgggtg cttgagagac actgggacactgtcagtgct 42000 agatttagca cagtattttg atctcgctag gtagaacact gctaataataatagctaata 42060 ataccttgtt ccaaatactg cttagcattt tgcatgtttt acttttatctaaagttttgt 42120 tttgttttat tatttattta tttatttatt ttgagacaga atctctctctgtcacccagg 42180 ctggagtgcc atggtgcgat cttggctcac tgcaacttta agcaattctcctgcctcagc 42240 ttcctgagta gctgggatta taggcgtgtg ccaccacgcc cagctactttctatattttt 42300 tgtagagatg gagtttcgcc atattggcca agctggtctc gaactcctgtcctcgaactc 42360 ctgtcctcaa gtgatccacc cgcctcagcc tctcaaagtg ctgggattacaggtgtgagc 42420 caccacaccc agcagtgttt tatttttgag acagggtatc attctgttgcccaggcttga 42480 gtgcagtggt gcaatcatag atcactgcag ccttttaact cctgggctcaagtcatcctc 42540 ctgcttagcc tcccaagtag ctaggaccac agacacatgc catcacacttggctattttt 42600 aaaaaatttt ttgtagagat ggggtctcgc tatgttaccc aaactggtcctgaactcctg 42660 gactcaattg atcctcccac cttggccttc caggtgctgg gatttctttgggagtacagc 42720 atggtacagc aggagatcat ttgatgttac ctctgtgcag tgttgctagtcagcgaaaga 42780 ctataatacc tgtggggaca gcgattagcc accacaacca gtctttatttaaagttatta 42840 aaaatggctg ggcgcagtgg ctcacacctg taatcctagc actttgggaggccgaggcag 42900 atggatcacc tgacgtgagg aatttgagac cagcctggcc aacatggtgaaaccccatct 42960 ctactaaaaa atacaaaaat tagctgggtg tggtcctgta gtcccagctacttgggaggc 43020 tggggcagga gaattacttg aacccaggag gcagaggttg cagtgagccgagattgtgcc 43080 actgcactcc agcctgggtg acagagagag attccatctc aaaaaaacaagttattaaaa 43140 atgtatatga atgctcctaa tatggtcagg aagcaaggaa gcgaaggatatattatgagt 43200 tttaagaagg tgcttagctg tatatttatc tttcaaaatg tattagaagattttagaatt 43260 ctttccttca tgtgccatct ctacaggcac ccatcagaaa aagcatactgccgttaccgt 43320 gaaactggtt gtaaaagaga aactatctat ttgcacctta aaagacagctagattttgct 43380 gattttcttc tttcggtttt ctttgtcagc aataatatgt gagaggacagattgttagat 43440 atgatagtat aaaaaatggt taatgacaat tcagaggcga ggagattctgtaaacttaaa 43500 attactataa atgaaattga tttgtcaaga ggataaattt tagaaaacacccaatacctt 43560 ataactgtct gttaatgctt gctttttctc tacctttctt ccttgtttcagttgggaagc 43620 ttttggctgc aagtaacaga aactcctaat tcaaatggct taagcaataaggaaatgtat 43680 attcccacat aactagacgt tcaaacaggc caggctccag cacttcagtacgtcaccagg 43740 gatctgggtt cttcccagct ctctgctctg ccatctttag cgctggcttcattctcagac 43800 tctggtagca tgatggctgt agctgtttca tgggcccctt caaacctcatagcaaccaga 43860 ggaagaaaat gagccatttt ttgagtctcc ttcatagact tgaataactctttttcagag 43920 cttctcacag caaacctctc ctcatgtctc ctcatgtctt attgttcagaaatgggtaat 43980 gtggccattt caccagtcac tgccaacaac aacgaggttc ctataattgtctctgagtaa 44040 ccctttggaa tggagagggt gttggtcagt ctacaaactg aacactgcagttctgcgctt 44100 tttaccagtg aaaaaatgta attattttcc cctcttaagg attaatattcttcaaatgta 44160 tgcctgttat ggatatagta tctttaaaat tttttatttt aatagctttaggggtacaca 44220 ctttttgctt acaggggtga attgtgtagt ggtgaagact cggcttttaatgtacttgtc 44280 acctgagtga tgtacattgt acccaatagg taatttttca tccattaccctccttccgcc 44340 ctcttccctt ctgagtctcc aacatccctt ataccactgt gtatgttcttgtgtacctac 44400 agctaagctt ccacttataa gtgagaacat gcagtatttg gttttccattcctgagttac 44460 ttcccttagg ataacagccc ccagttccgt ccaagttgct gcaaaatacattattcttct 44520 ttatggctga gtaatagtcc atggtacata tataccacat tttctttatccacttatcag 44580 ttgatggaca cttaggttaa ttccattcaa tttcattcaa tttaagtatatttgtaagga 44640 gctaaagctg aaaattaaat tttagatctt tcaatactct taaattttatatgtaagtgg 44700 tttttatatt ttcacatttg aaataaagta atttttataa ccttgatattgtatgactat 44760 tcttttagta atgtaaagcc tacagactcc tacatttgga accactagtgtgttgtttca 44820 ccccttgtta tactatcagg atcctcga 44848 43 2396 DNA Musmusculus 43 tttctagttg cttttagcca atgtcggatc aggtttttca agcgacaaagagatactgag 60 atcctgggca gaggacatcc tagctcggtc agatttgggc aggctcaagtgaccagtgtc 120 ttaaggcaga agggagtcgg ggtagggtct ggctgaaccc tcaaccggggcttttaactc 180 agggtctagt cctggcgcca aatggatggg acctagaaaa ggtgacagagtgcgcaggac 240 accaggaagc tggtcccacc cctgcgcggc tcccgggcgc tccctccccaggcctccgag 300 gatcttggat tctggccacc tccgcaccct ttggatgggt gtggatgatttcaaaagtgg 360 acgtgaccgc ggcggagggg aaagccagca cggaaatgaa agagagcgaggaggggaggg 420 cggggagggg agggcgctag ggagggactc ccgggagggg tgggagggatggagcgctgt 480 gggagggtac tgagtcctgg cgccagaggc gaagcaggac cggttgcagggggcttgagc 540 cagcgcgccg gctgccccag ctctcccggc agcgggcggt ccagccaggtgggatgctga 600 ggctgctgct gctgtggctc tgggggccgc tcggtgccct ggcccagggcgcccccgcgg 660 ggaccgcgcc gaccgacgac gtggtagact tggagtttta caccaagcggccgctccgaa 720 gcgtgagtcc ctcgttcctg tccatcacca tcgacgccag cctggccaccgacccgcgct 780 tcctcacctt cctgggctct ccaaggctcc gtgctctggc tagaggcttatctcctgcat 840 acttgagatt tggcggcaca aagactgact tccttatttt tgatccggacaaggaaccga 900 cttccgaaga aagaagttac tggaaatctc aagtcaacca tgatatttgcaggtctgagc 960 cggtctctgc tgcggtgttg aggaaactcc aggtggaatg gcccttccaggagctgttgc 1020 tgctccgaga gcagtaccaa aaggagttca agaacagcac ctactcaagaagctcagtgg 1080 acatgctcta cagttttgcc aagtgctcgg ggttagacct gatctttggtctaaatgcgt 1140 tactacgaac cccagactta cggtggaaca gctccaacgc ccagcttctccttgactact 1200 gctcttccaa gggttataac atctcctggg aactgggcaa tgagcccaacagtttctgga 1260 agaaagctca cattctcatc gatgggttgc agttaggaga agactttgtggagttgcata 1320 aacttctaca aaggtcagct ttccaaaatg caaaactcta tggtcctgacatcggtcagc 1380 ctcgagggaa gacagttaaa ctgctgagga gtttcctgaa ggctggcggagaagtgatcg 1440 actctcttac atggcatcac tattacttga atggacgcat cgctaccaaagaagattttc 1500 tgagctctga tgcgctggac acttttattc tctctgtgca aaaaattctgaaggtcacta 1560 aagagatcac acctggcaag aaggtctggt tgggagagac gagctcagcttacggtggcg 1620 gtgcaccctt gctgtccaac acctttgcag ctggctttat gtggctggataaattgggcc 1680 tgtcagccca gatgggcata gaagtcgtga tgaggcaggt gttcttcggagcaggcaact 1740 accacttagt ggatgaaaac tttgagcctt tacctgatta ctggctctctcttctgttca 1800 agaaactggt aggtcccagg gtgttactgt caagagtgaa aggcccagacaggagcaaac 1860 tccgagtgta tctccactgc actaacgtct atcacccacg atatcaggaaggagatctaa 1920 ctctgtatgt cctgaacctc cataatgtca ccaagcactt gaaggtaccgcctccgttgt 1980 tcaggaaacc agtggatacg taccttctga agccttcggg gccggatggattactttcca 2040 aatctgtcca actgaacggt caaattctga agatggtgga tgagcagaccctgccagctt 2100 tgacagaaaa acctctcccc gcaggaagtg cactaagcct gcctgccttttcctatggtt 2160 tttttgtcat aagaaatgcc aaaatcgctg cttgtatatg aaaataaaaggcatacggta 2220 cccctgagac aaaagccgag gggggtgtta ttcataaaac aaaaccctagtttaggaggc 2280 cacctccttg ccgagttcca gagcttcggg agggtggggt acacttcagtattacattca 2340 gtgtggtgtt ctctctaaga agaatactgc aggtggtgac agttaatagcactgtg 2396 44 535 PRT Mus musculus 44 Met Leu Arg Leu Leu Leu Leu TrpLeu Trp Gly Pro Leu Gly Ala Leu 1 5 10 15 Ala Gln Gly Ala Pro Ala GlyThr Ala Pro Thr Asp Asp Val Val Asp 20 25 30 Leu Glu Phe Tyr Thr Lys ArgPro Leu Arg Ser Val Ser Pro Ser Phe 35 40 45 Leu Ser Ile Thr Ile Asp AlaSer Leu Ala Thr Asp Pro Arg Phe Leu 50 55 60 Thr Phe Leu Gly Ser Pro ArgLeu Arg Ala Leu Ala Arg Gly Leu Ser 65 70 75 80 Pro Ala Tyr Leu Arg PheGly Gly Thr Lys Thr Asp Phe Leu Ile Phe 85 90 95 Asp Pro Asp Lys Glu ProThr Ser Glu Glu Arg Ser Tyr Trp Lys Ser 100 105 110 Gln Val Asn His AspIle Cys Arg Ser Glu Pro Val Ser Ala Ala Val 115 120 125 Leu Arg Lys LeuGln Val Glu Trp Pro Phe Gln Glu Leu Leu Leu Leu 130 135 140 Arg Glu GlnTyr Gln Lys Glu Phe Lys Asn Ser Thr Tyr Ser Arg Ser 145 150 155 160 SerVal Asp Met Leu Tyr Ser Phe Ala Lys Cys Ser Gly Leu Asp Leu 165 170 175Ile Phe Gly Leu Asn Ala Leu Leu Arg Thr Pro Asp Leu Arg Trp Asn 180 185190 Ser Ser Asn Ala Gln Leu Leu Leu Asp Tyr Cys Ser Ser Lys Gly Tyr 195200 205 Asn Ile Ser Trp Glu Leu Gly Asn Glu Pro Asn Ser Phe Trp Lys Lys210 215 220 Ala His Ile Leu Ile Asp Gly Leu Gln Leu Gly Glu Asp Phe ValGlu 225 230 235 240 Leu His Lys Leu Leu Gln Arg Ser Ala Phe Gln Asn AlaLys Leu Tyr 245 250 255 Gly Pro Asp Ile Gly Gln Pro Arg Gly Lys Thr ValLys Leu Leu Arg 260 265 270 Ser Phe Leu Lys Ala Gly Gly Glu Val Ile AspSer Leu Thr Trp His 275 280 285 His Tyr Tyr Leu Asn Gly Arg Ile Ala ThrLys Glu Asp Phe Leu Ser 290 295 300 Ser Asp Ala Leu Asp Thr Phe Ile LeuSer Val Gln Lys Ile Leu Lys 305 310 315 320 Val Thr Lys Glu Ile Thr ProGly Lys Lys Val Trp Leu Gly Glu Thr 325 330 335 Ser Ser Ala Tyr Gly GlyGly Ala Pro Leu Leu Ser Asn Thr Phe Ala 340 345 350 Ala Gly Phe Met TrpLeu Asp Lys Leu Gly Leu Ser Ala Gln Met Gly 355 360 365 Ile Glu Val ValMet Arg Gln Val Phe Phe Gly Ala Gly Asn Tyr His 370 375 380 Leu Val AspGlu Asn Phe Glu Pro Leu Pro Asp Tyr Trp Leu Ser Leu 385 390 395 400 LeuPhe Lys Lys Leu Val Gly Pro Arg Val Leu Leu Ser Arg Val Lys 405 410 415Gly Pro Asp Arg Ser Lys Leu Arg Val Tyr Leu His Cys Thr Asn Val 420 425430 Tyr His Pro Arg Tyr Gln Glu Gly Asp Leu Thr Leu Tyr Val Leu Asn 435440 445 Leu His Asn Val Thr Lys His Leu Lys Val Pro Pro Pro Leu Phe Arg450 455 460 Lys Pro Val Asp Thr Tyr Leu Leu Lys Pro Ser Gly Pro Asp GlyLeu 465 470 475 480 Leu Ser Lys Ser Val Gln Leu Asn Gly Gln Ile Leu LysMet Val Asp 485 490 495 Glu Gln Thr Leu Pro Ala Leu Thr Glu Lys Pro LeuPro Ala Gly Ser 500 505 510 Ala Leu Ser Leu Pro Ala Phe Ser Tyr Gly PhePhe Val Ile Arg Asn 515 520 525 Ala Lys Ile Ala Ala Cys Ile 530 535 452396 DNA Mus musculus CDS (594)..(2198) 45 tttctagttg cttttagccaatgtcggatc aggtttttca agcgacaaag agatactgag 60 atcctgggca gaggacatcctagctcggtc agatttgggc aggctcaagt gaccagtgtc 120 ttaaggcaga agggagtcggggtagggtct ggctgaaccc tcaaccgggg cttttaactc 180 agggtctagt cctggcgccaaatggatggg acctagaaaa ggtgacagag tgcgcaggac 240 accaggaagc tggtcccacccctgcgcggc tcccgggcgc tccctcccca ggcctccgag 300 gatcttggat tctggccacctccgcaccct ttggatgggt gtggatgatt tcaaaagtgg 360 acgtgaccgc ggcggaggggaaagccagca cggaaatgaa agagagcgag gaggggaggg 420 cggggagggg agggcgctagggagggactc ccgggagggg tgggagggat ggagcgctgt 480 gggagggtac tgagtcctggcgccagaggc gaagcaggac cggttgcagg gggcttgagc 540 cagcgcgccg gctgccccagctctcccggc agcgggcggt ccagccaggt ggg atg 596 Met 1 ctg agg ctg ctg ctgctg tgg ctc tgg ggg ccg ctc ggt gcc ctg gcc 644 Leu Arg Leu Leu Leu LeuTrp Leu Trp Gly Pro Leu Gly Ala Leu Ala 5 10 15 cag ggc gcc ccc gcg gggacc gcg ccg acc gac gac gtg gta gac ttg 692 Gln Gly Ala Pro Ala Gly ThrAla Pro Thr Asp Asp Val Val Asp Leu 20 25 30 gag ttt tac acc aag cgg ccgctc cga agc gtg agt ccc tcg ttc ctg 740 Glu Phe Tyr Thr Lys Arg Pro LeuArg Ser Val Ser Pro Ser Phe Leu 35 40 45 tcc atc acc atc gac gcc agc ctggcc acc gac ccg cgc ttc ctc acc 788 Ser Ile Thr Ile Asp Ala Ser Leu AlaThr Asp Pro Arg Phe Leu Thr 50 55 60 65 ttc ctg ggc tct cca agg ctc cgtgct ctg gct aga ggc tta tct cct 836 Phe Leu Gly Ser Pro Arg Leu Arg AlaLeu Ala Arg Gly Leu Ser Pro 70 75 80 gca tac ttg aga ttt ggc ggc aca aagact gac ttc ctt att ttt gat 884 Ala Tyr Leu Arg Phe Gly Gly Thr Lys ThrAsp Phe Leu Ile Phe Asp 85 90 95 ccg gac aag gaa ccg act tcc gaa gaa agaagt tac tgg aaa tct caa 932 Pro Asp Lys Glu Pro Thr Ser Glu Glu Arg SerTyr Trp Lys Ser Gln 100 105 110 gtc aac cat gat att tgc agg tct gag ccggtc tct gct gcg gtg ttg 980 Val Asn His Asp Ile Cys Arg Ser Glu Pro ValSer Ala Ala Val Leu 115 120 125 agg aaa ctc cag gtg gaa tgg ccc ttc caggag ctg ttg ctg ctc cga 1028 Arg Lys Leu Gln Val Glu Trp Pro Phe Gln GluLeu Leu Leu Leu Arg 130 135 140 145 gag cag tac caa aag gag ttc aag aacagc acc tac tca aga agc tca 1076 Glu Gln Tyr Gln Lys Glu Phe Lys Asn SerThr Tyr Ser Arg Ser Ser 150 155 160 gtg gac atg ctc tac agt ttt gcc aagtgc tcg ggg tta gac ctg atc 1124 Val Asp Met Leu Tyr Ser Phe Ala Lys CysSer Gly Leu Asp Leu Ile 165 170 175 ttt ggt cta aat gcg tta cta cga acccca gac tta cgg tgg aac agc 1172 Phe Gly Leu Asn Ala Leu Leu Arg Thr ProAsp Leu Arg Trp Asn Ser 180 185 190 tcc aac gcc cag ctt ctc ctt gac tactgc tct tcc aag ggt tat aac 1220 Ser Asn Ala Gln Leu Leu Leu Asp Tyr CysSer Ser Lys Gly Tyr Asn 195 200 205 atc tcc tgg gaa ctg ggc aat gag cccaac agt ttc tgg aag aaa gct 1268 Ile Ser Trp Glu Leu Gly Asn Glu Pro AsnSer Phe Trp Lys Lys Ala 210 215 220 225 cac att ctc atc gat ggg ttg cagtta gga gaa gac ttt gtg gag ttg 1316 His Ile Leu Ile Asp Gly Leu Gln LeuGly Glu Asp Phe Val Glu Leu 230 235 240 cat aaa ctt cta caa agg tca gctttc caa aat gca aaa ctc tat ggt 1364 His Lys Leu Leu Gln Arg Ser Ala PheGln Asn Ala Lys Leu Tyr Gly 245 250 255 cct gac atc ggt cag cct cga gggaag aca gtt aaa ctg ctg agg agt 1412 Pro Asp Ile Gly Gln Pro Arg Gly LysThr Val Lys Leu Leu Arg Ser 260 265 270 ttc ctg aag gct ggc gga gaa gtgatc gac tct ctt aca tgg cat cac 1460 Phe Leu Lys Ala Gly Gly Glu Val IleAsp Ser Leu Thr Trp His His 275 280 285 tat tac ttg aat gga cgc atc gctacc aaa gaa gat ttt ctg agc tct 1508 Tyr Tyr Leu Asn Gly Arg Ile Ala ThrLys Glu Asp Phe Leu Ser Ser 290 295 300 305 gat gcg ctg gac act ttt attctc tct gtg caa aaa att ctg aag gtc 1556 Asp Ala Leu Asp Thr Phe Ile LeuSer Val Gln Lys Ile Leu Lys Val 310 315 320 act aaa gag atc aca cct ggcaag aag gtc tgg ttg gga gag acg agc 1604 Thr Lys Glu Ile Thr Pro Gly LysLys Val Trp Leu Gly Glu Thr Ser 325 330 335 tca gct tac ggt ggc ggt gcaccc ttg ctg tcc aac acc ttt gca gct 1652 Ser Ala Tyr Gly Gly Gly Ala ProLeu Leu Ser Asn Thr Phe Ala Ala 340 345 350 ggc ttt atg tgg ctg gat aaattg ggc ctg tca gcc cag atg ggc ata 1700 Gly Phe Met Trp Leu Asp Lys LeuGly Leu Ser Ala Gln Met Gly Ile 355 360 365 gaa gtc gtg atg agg cag gtgttc ttc gga gca ggc aac tac cac tta 1748 Glu Val Val Met Arg Gln Val PhePhe Gly Ala Gly Asn Tyr His Leu 370 375 380 385 gtg gat gaa aac ttt gagcct tta cct gat tac tgg ctc tct ctt ctg 1796 Val Asp Glu Asn Phe Glu ProLeu Pro Asp Tyr Trp Leu Ser Leu Leu 390 395 400 ttc aag aaa ctg gta ggtccc agg gtg tta ctg tca aga gtg aaa ggc 1844 Phe Lys Lys Leu Val Gly ProArg Val Leu Leu Ser Arg Val Lys Gly 405 410 415 cca gac agg agc aaa ctccga gtg tat ctc cac tgc act aac gtc tat 1892 Pro Asp Arg Ser Lys Leu ArgVal Tyr Leu His Cys Thr Asn Val Tyr 420 425 430 cac cca cga tat cag gaagga gat cta act ctg tat gtc ctg aac ctc 1940 His Pro Arg Tyr Gln Glu GlyAsp Leu Thr Leu Tyr Val Leu Asn Leu 435 440 445 cat aat gtc acc aag cacttg aag gta ccg cct ccg ttg ttc agg aaa 1988 His Asn Val Thr Lys His LeuLys Val Pro Pro Pro Leu Phe Arg Lys 450 455 460 465 cca gtg gat acg tacctt ctg aag cct tcg ggg ccg gat gga tta ctt 2036 Pro Val Asp Thr Tyr LeuLeu Lys Pro Ser Gly Pro Asp Gly Leu Leu 470 475 480 tcc aaa tct gtc caactg aac ggt caa att ctg aag atg gtg gat gag 2084 Ser Lys Ser Val Gln LeuAsn Gly Gln Ile Leu Lys Met Val Asp Glu 485 490 495 cag acc ctg cca gctttg aca gaa aaa cct ctc ccc gca gga agt gca 2132 Gln Thr Leu Pro Ala LeuThr Glu Lys Pro Leu Pro Ala Gly Ser Ala 500 505 510 cta agc ctg cct gccttt tcc tat ggt ttt ttt gtc ata aga aat gcc 2180 Leu Ser Leu Pro Ala PheSer Tyr Gly Phe Phe Val Ile Arg Asn Ala 515 520 525 aaa atc gct gct tgtata tgaaaataaa aggcatacgg tacccctgag 2228 Lys Ile Ala Ala Cys Ile 530535 acaaaagccg aggggggtgt tattcataaa acaaaaccct agtttaggag gccacctcct2288 tgccgagttc cagagcttcg ggagggtggg gtacacttca gtattacatt cagtgtggtg2348 ttctctctaa gaagaatact gcaggtggtg acagttaata gcactgtg 2396 46 385DNA Rattus norvegicus 46 cggccgctgc tgctgctgtg gctctggggg cggctccgtgccctgaccca aggcactccg 60 gcggggaccg cgccgaccaa agacgtggtg gacttggagttttacaccaa gaggctattc 120 caaagcgtga gtccctcgtt cctgtccatc accatcgacgccagtctggc caccgaccct 180 cggttcctca ccttcctgag ctctccacgg cttcgagccctgtctagagg cttatctcct 240 gcgtacttga gatttggcgg caccaagact gacttccttatttttgatcc caacaacgaa 300 cccacctctg aagaaagaag ttactggcaa tctcaagacaacaatgatat ttgcgggtct 360 gaccgggtct ccgctgacgt gttga 385 47 541 DNARattus norvegicus misc_feature (507)..(507) Any nucleotide 47 aaatcaggacatatccttca cttatttgcc tcttggtcat attggaggca tttgtattca 60 tttttaataaccctcaaaat agtgcatgca aagtgctaag cgtcatttgc cacatggtgc 120 cattaactgtcaccacctgc agtggtctac ttagagaaca ccgcactgga tgttaacact 180 gaagcgcgtgccccgccctc ccgaggctct ggatccagcg ttgaagcttg ccccgccctc 240 ccgaggctctggatccagca ctggagcatg ccccgccctc ccgaggctct ggagcttgct 300 aaggagtccgctccctaccg ctggggtttt gctttattct tatgaatgac acccctgacc 360 gctttcgtctcaggggtact gtaatgcctt ttattttcat atacaagctg cgattttggc 420 atttcttatgacaaaaaacc cataggaaaa ggcgggcacg cttagtgagc ttcctgcggg 480 gagaggtttttctgttagag ctggcanggt ctgctcatcg accatcttca ggcctcgtgc 540 c 541 48 17DNA Artificial sequence Single strand DNA oligonucleotide 48 ataggcagctgacctga 17 49 24 DNA Artificial sequence Single strand DNAoligonucleotide 49 tgacttgaga ttgccagtaa cttc 24 50 24 DNA Artificialsequence Single strand DNA oligonucleotide 50 ctgtccaact caatggtcta actc24 51 20 DNA Artificial sequence Single strand DNA oligonucleotide 51tctagagcct ctgctaacca 20

What is claimed is:
 1. A transgenic non-human animal whose genomecomprises an exogenous polynucleotide sequence integrated into saidgenome, said exogenous polynucleotide sequence including a promoteractive in tissues of the non-human, and a region encoding a humanheparanase, wherein said promoter and said region encoding humanheparanase are operably linked in said exogenous polynucleotide suchthat human heparanase is expressed in at least a portion of the cells ofthe non-human animal.
 2. The transgenic non-human animal of claim 1,being homozygous for said exogenous polynucleotide sequence.
 3. Thetransgenic non-human animal of claim 1, being heterozygous for saidexogenous polynucleotide sequence.
 4. The transgenic non-human animal ofclaim 1, having a single locus harboring said exogenous polynucleotidesequence.
 5. The transgenic non-human animal of claim 1, having at leasttwo loci each harboring said exogenous polynucleotide sequence.
 6. Thetransgenic non-human animal of claim 1, wherein said human heparanase isgenetically modified to be cleavable into an active form via a protease.7. The transgenic non-human animal of claim 1, wherein said heparanaseis processed by an endogenous protease of the non-human animal into anactive form.
 8. The transgenic non-human animal of claim 1, wherein saidregion of said exogenous polynucleotide sequence encodes an active formof heparanase.
 9. The transgenic non-human animal of claim 1, being amammal.
 10. The transgenic non-human animal of claim 1, being an avian.11. The transgenic non-human animal of claim 1, wherein said exogenouspolynucleotide sequence includes a tissue specific promoter fordirecting expression of said heparanase in a tissue specific manner. 12.The transgenic non-human animal of claim 1, wherein said promoter is aconstitutive promoter for directing expression of said heparanase inconstitutive manner.
 13. The transgenic non-human animal of claim 1,wherein said promoter is an inducible promoter for directing expressionof said heparanase in an inducible manner.
 14. The transgenic non-humananimal of claim 1, wherein said promoter is selected from the groupconsisting of beta-lactoglobulin promoter, Rb promoter,preproendothelin-1 promoter, beta-actin promoter, TetO promoter,metallothionein promoter, whey acidic protein (WAP) promoter, caseinpromoter and lactalbumin promoter.
 15. The transgenic non-human mammalof claim 9, wherein said heparanase is expressed in, and secreted by,cells of mammary glands of said mammal.
 16. The transgenic avian ofclaim 10, wherein said promoter is selected from the group consisting ofchicken lyzozyme promoter, cytomegalovirus promoter and chickenimmunoglobulin promoter.
 17. The transgenic non-human avian of claim 10,wherein said heparanase is expressed in, and secreted by, egg producingcells of said avian.
 18. Sex cells derived from the transgenic non-humananimal of claim
 1. 19. Semen derived from the transgenic non-humananimal of claim
 1. 20. An embryo derived from the transgenic non-humananimal of claim
 1. 21. A composition of matter comprising milk derivedfrom a non-human transgenic mammal, said milk having detectable humanheparanase activity.
 22. A composition of matter comprising egg yolkand/or white from a transgenic avian, said egg yolk and/or white havingdetectable human heparanase activity.
 23. A method of producingrecombinant human heparanase, the method comprising the steps of: (a)obtaining a transgenic non-human mammal having mammary glands, whosegenome comprises an exogenous polynucleotide sequence integrated intosaid genome, said exogenous polynucleotide sequence including a promoteractive in tissues of the non-human mammal, and a region encoding a humanheparanase, wherein said promoter and said region encoding humanheparanase are operably linked in said exogenous polynucleotide suchthat the recombinant human heparanase is secreted into milk beingproduced by said mammary glands; (b) milking said non-human mammal so asto obtain milk containing the recombinant human heparanase; and (c)purifying the recombinant human heparanase from said milk.
 24. Themethod of claim 23, wherein said promoter active in tissues of saidnon-human mammal is a milk protein gene promoter.
 25. The method ofclaim 24, wherein said milk protein gene promoter is selected from thegroup consisting of beta-lactoglobulin promoter, Rb promoter,preproendothelin-1 promoter, whey acidic protein (WAP) promoter, caseinpromoter and lactalbumin promoter.
 26. A method of producing recombinanthuman heparanase, the method comprising the steps of: (a) obtaining atransgenic female avian having egg producing cells whose genomecomprises an exogenous polynucleotide sequence integrated into saidgenome, said exogenous polynucleotide sequence including a promoteractive in tissues of said transgenic female avian, and a region encodinga human heparanase, wherein said promoter and said region encoding humanheparanase are operably linked in said exogenous polynucleotide suchthat the recombinant human heparanase is secreted into eggs beingproduced by said egg producing cells; (b) collecting eggs laid by saidtransgenic female avian so as to obtain eggs containing the recombinanthuman heparanase; and (c) purifying the recombinant human heparanasefrom said eggs.
 27. The method of claim 26, wherein said promoter activein tissues of said transgenic female avian is an egg protein genepromoter.
 28. The method of claim 27, wherein said egg protein genepromoter is selected from the group consisting of chicken lyzozymepromoter and chicken immunoglobulin promoter.